UNIVERSITATEA DE ȘTIINȚE AGRICOLE ȘI MEDICINĂ VETERINARĂ “ION IONESCU DE LA BRAD” IAȘI
LUCRĂRI ȘTIINȚIFICE
VOL. 60 MEDICINĂ VETERINARĂ
PARTEA a 3-a
EDITURA “ION IONESCU DE LA BRAD” IAȘI 2017
Coordonatorii Revistei Redactor responsabil: Prof. dr. Vasile VÎNTU - USAMV Iaşi Redactor adjunct: Prof. dr. Liviu-Dan MIRON - USAMV Iaşi Membri: - Prof. dr. Costel SAMUIL - USAMV Iaşi - Prof. dr. Lucia DRAGHIA - USAMV Iaşi - Prof. dr. Gheorghe SAVUȚA - USAMV Iaşi - Prof. dr. Paul-Corneliu BOIȘTEANU - USAMV Iaşi
Colegiul de Redacţie al Seriei "Medicină veterinară" Redactor şef: Prof. dr. Gheorghe SAVUȚA - USAMV Iaşi Redactor adjunct: Prof. dr. Mihai MAREŞ - USAMV Iaşi Membri: Prof. dr. Gheorghe SOLCAN - USAMV Iaşi Prof. dr. Gheorghe DRUGOCIU - USAMV Iaşi Conf. dr. Geta PAVEL - USAMV Iaşi Conf. dr. Viorel Cezar FLORIȘTEAN - USAMV Iaşi Conf. dr. Valentin NĂSTASĂ - USAMV Iaşi Asist. dr. Mariana GRECU
Referenţi ştiinţifici: Prof. dr. Abdelfatah NOUR - Purdue University, SUA Prof. dr. Gheorghe SAVUŢA - USAMV Iaşi Prof. dr. Liviu MIRON - USAMV Iaşi Prof. dr. Gheorghe SOLCAN - USAMV Iaşi Acad. Ion TODERAŞ - Zoology Institute, Chisinau, Republica Moldova Assoc. Prof. Dorina CARTER - University of Liverpool, UK Prof. dr. Elena VELESCU - USAMV Iaşi Prof. dr. Gheorghe DRUGOCIU - USAMV Iaşi Prof. dr. Vasile VULPE - USAMV Iaşi Prof. dr. Cornel CĂTOI - USAMV Cluj-Napoca Prof. dr. Gabriel PREDOI - USAMV Bucureşti Prof. dr. Viorel HERMAN - USAMVB Timişoara Prof. dr. Mihai MAREȘ - USAMV Iași Conf. dr. Valentin NĂSTASĂ - USAMV Iaşi Conf. dr. Sorin-Aurelian PAŞCA - USAMV Iaşi
on -line ISSN 2393 – 4603 ISSN–L 1454 – 7406
CONTENTS
The effect of habitat on hair copper, molybdenum, and selenium
levels in cats
Gheorghe Valentin Goran, Emanuela Badea, Cristina Țoca, Victor
Crivineanu
325 - 329
Copper-releasing, borate-based glasses with antibacterial properties:
synthesis and in vitro characterization
Cristina Lelescu, Aurel Muste, Marian Taulescu, Gheorghe Borodi, Marin
Șenilă, Lucian Barbu-Tudoran, Răzvan Ștefan
330 - 337
Searches on the application of a method of induction and syncronization
of estrus in cows postpartum based on two doses of GnRH and
prostaglandin F2 α, with programmed insemination
Elena Ruginosu, S.I Borș, Ș. Creangă, D.L. Dascălu, Mădălina Alexandra
Davidescu
338 - 345
vagin*l smear, progesterone levels, and ultrasound examination of the
ovaries as methods of determining the moment of ovulation in bitches
comparative study
G. Otavă, C. Mircu, Violeta Igna, Simona Marc Zarcula, D. Lo Presti
346 - 353
Effect of rozmarinic acid supplementation on in vitro maturation
of bovine oocytes
Simona Marc, Camelia Tulcan, Oana Boldura, A. Solonar, G. Otavă,
G. Godja, I. Huțu, C. Mircu
354 - 358
Evaluation of sows oocytes viability through Trypan Blue staining after
vitrification
Simona Marc, C. Mircu, Nicoleta Crețan, G. Otavă, Camelia Tulcan, I. Huțu
359 - 364
Content of amino acids in blood serum in sows with idiopathic
hipogalaxy
Viorica Gurdis
365 - 368
Cell growth characteristics of equine synovial fluid stem cells
Emoke Pall, Klementina Katalin Pall, Cristian Crecan, Simona Ciupe, Mihai
Cenariu, Ioan Groza
369 - 373
Epidemiology of atopic dermatitis and other allergic skin diseases in
dogs and cats in Western Romania
Tiana Suici, Gh. Darabus, Narcisa Mederle, Mirela Imre, C. Sirbu, S. Morariu
374 - 377
Generating bovine embryos through ICSI
Thomas Keller, Simona Marc, Horia Cernescu, Camelia Tulcan, Ioan Huțu,
Gabriel Otava, Ana-Maria Rațiu, Georgiana Ungureanu, Călin Mircu
378 - 385
The behaviour pattern of several gastrointestinal nematode genera in
sheep and cattle from bethausen, Timis County
C. Sîrbu, Gh. Darabus, M. Ilie, Mirela Imre, Tiana Suici, S. Morariu
386 - 391
Preliminary research regarding the prevalence of digestive and
respiratory parasitosis in meat cattle from the Hârtibaci Valley,
Sibiu County
Radu Nechiti, Gheorghe Dărăbuș, Sorin Morariu
392 - 396
Research on metabolic status in periparturient cows
Sorin D. Sorescu, Carmen Ioniță, Alice Grigore, Emilia Balint, Aana Maria
Goanță, Roxana Țîmpău, Lucian Ioniță
397 - 402
New Zealand Crossbred male rabbitproduction performance fed with
fructooligosacharide prebiotic isolated from banana peel
Suraya Kaffi Syahpura, Kusmajadi Suradi, Husmy Yurmiati, Diding
Latifudin
403 - 411
325
The effect of habitat on hair copper, molybdenum, and selenium
levels in cats
Gheorghe Valentin GORAN1, Emanuela BADEA1, Cristina ȚOCA2, Victor CRIVINEANU1 1 Faculty of Veterinary Medicine, UASVM of Bucharest, 105 Splaiul Independentei, 050097,
5th district, Bucharest, Romania, EU; 2IDAH of Bucharest, 63 Dr. Staicovici, 050557, 5th district, Bucharest, Romania, EU
[emailprotected]
Abstract
Cu, Mo, and Se are essential trace minerals, which maintain proper activity of some animal
organisms functions. The main goal of this study was the assessment of Cu, Mo and Se levels in the hair of
pet cats in an urban environment. The hair samples were collected from flank region from 20 clinically
healthy pet cats. Analysis of hair Cu, Mo, and Se content of pet cats kept indoors (5 males and 5 females)
and outdoors (5 males and 5 females), were performed by inductively coupled plasma mass spectrometry
(ICP-MS). The mean Cu level in indoor pet cats was 19.47 mg•kg−1 for males and 10.58 mg•kg−1 for females,
and in outdoor male cats was 10.33 mg•kg−1 and 14.32 mg•kg−1 for females. Generally, Mo registered lower
mean levels when mean Cu levels were higher and higher levels when Cu was lower, indifferent of habitat,
sex or age. The mean Se hair levels registered insignificant differences for the same habitat in pet cats below
5 years and above 5 years. In this study, the habitat statistically insignificant influenced hair Cu, Mo, and
Se levels in pet cats.
Key words: habitat, hair, cat, copper, molybdenum, selenium
Introduction
In classification proposed by Frieden (1985), Cu, Mo, and Se are essential trace minerals,
which maintain proper activity of some animal organisms functions, and deficiency in this elements
leads to disorders and may prove fatal (Prashanth ET AL., 2015).
Cu is the key trace element for enzymes necessary for increasing the strength of keratin
fiber, and has very important role in activity of essential enzyme systems implicated in formation,
growth or repair of keratin-rich tissues. (Goluch-Koniuszy, 2016) Cu favors the intestinal
absorption of iron and its incorporation into hemoglobin and is a component of many enzymes. Cu
plays an important role in reducing cellular lesions caused by free radicals. Cu is also involved in
the synthesis of collagen in tendons and myelin in the nervous system. Cu also participates in the
synthesis of melanin, which is a hair pigment. (Collins, 2014) The Cu deficit most likely induced
by zinc, through a mechanism that can be explained by the competitive absorption of zinc and Cu
in enterocytes in the small intestine. Excessive zinc intake stimulates the production of
metallothionein. However, Cu has a higher affinity for metallothionein, eliminating zinc, and then
excreting, which leads to hypocupremia. (Green and Weaver, 2008)
Mo concentrates in the liver, kidney, bone and significant amounts are found in the dental
enamel and hair, and has an essential role as cofactor of some important enzyme systems – xanthine
oxidase, an enzyme involved in the formation of uric acid; aldehyde oxidase, an enzyme involved
in detoxification (Mendel, 2013a), and is involved in hair health by inducing secondary Cu
deficiency (Phoon et al., 2011). Mo has been shown in animals to be involved with fat, purine and
sulfate metabolism, and it is also involved in detoxification and intimately involved in Cu
metabolism. Symptoms associated with a Mo deficiency are represented by impaired growth, tooth
decay, male impotence, xanthine stones, and also symptoms of Cu toxicity. (Mendel, 2013b) Mo
excess could determine acute toxicity, which result in severe diarrhea, and chronic toxicity, which
may cause gout. Cu deficiency symptoms may also occur, including skin problems, hair loss,
326
growth retardation, osteoporosis, thyroid abnormality, bone and joint abnormalities and weight
loss. (Barceloux and Barceloux, 1999) Mo and sulfur also antagonize Cu by a ternary interaction
involving the formation of Cu thiomolybdate, which results in reduced Cu absorption. Tungsten is
a Mo antagonist in several oxidative enzymes that require Mo (e.g. xanthine oxidase). (Frieden,
1985)
Se is an essential component of Se proteins, which play an important role in many
biological functions, such as antioxidant defense, thyroid hormone formation, DNA synthesis,
fertility and reproduction (Mehdi et al., 2013; Sunde, 2014). Hair gets trace elements especially
from the blood and is able to integrate Se into its matrix during keratinization. Se is part of at least
35 proteins, many of which are enzymes, and Se deficiency causes hair loss and pseudo albinism
(Masumoto et al., 2007). Although much less common than Se deficiency, Se toxicity can affect
individuals as a result of excessive supplementation, causing hair loss. (Fairweather-Tait et al.,
2011) Hair can be an excellent sample for assessing mineral status, especially of trace elements,
in the organism, because their hair concentration is usually higher than in the blood. (Foo et al.,
1993; Skibniewska et al., 2011; Skibniewska et al., 2013; Skibniewski et al., 2013)
Animal hair can be a biomarker of environmental pollution, and also for assessing animal
mineral status. In recent years, as a bioindicator of metal pollution or different organ diseases, hair
samples from domestic and wild species such as dog, cat, cattle, horse, goat, sheep, camel,
European bison, elk, brown bear, wolf, fox, wild boar, squirrel and seal. (Combs, 1987; Medvedev,
1999; Liu, 2003; Ikemoto et al., 2004; Rashed and Soltan, 2005; Hawkins and Ragnarsdottir, 2009;
Crivineanu et al., 2010; Skibniewski et al., 2010; Filistowicz et al., 2011; de Almeida Curi et al.,
2012; Hernández-Moreno et al., 2013; Badea et al., 2016a; Badea et al., 2016b; Goran et al., 2017)
The main goal of this study was the assessment of Cu, Mo and Se levels in the hair of pet
cats in an urban environment, using inductively coupled plasma mass spectrometry (ICP-MS).
Materials and methods
Sampling and samples preparation - The hair samples were collected from flank region
from 20 clinically healthy pet cats from an urban environment. Analysis of hair Cu, Mo, and Se
content of pet cats kept indoors (5 males and 5 females) and outdoors (5 males and 5 females),
were performed by inductively coupled plasma mass spectrometry (ICP-MS).
Before analysis the hair samples (n=20) were weighed (approximately 0.5 g) using an
analytic balance (precision 4 decimals) and placed in individual PPR vials with a capacity of 14
mL. Disintegration of the organic matter was done by cold wet mineralization, adding 5 mL of
nitric acid 65% suprapur (d=1.39) and 1 mL of hydrochloric acid 30% suprapur (d=1.15) over the
hair samples. The samples were disintegrated at room temperature into the fume hood for one week.
Spectrometric analysis - Digested samples were diluted to 10 mL with ultrapure water and
analyzed by Perkin-Elmer Elan DRC II ICP–MS spectrometer (RF1100 W; reading time 30 s,
washing time 30 s, nebulizer gas flow 0.5 L•min-1; auxiliary gas flow 0.5 L•min-1; sample injection
pump flow 50 rpm). Calibration curves were developed using standard solutions of 0.001 ppm,
0.05 ppm, 0.01 ppm, 0.025 ppm, and 0.05 ppm (for low concentrations of minerals as Se), and 0.1
ppm, 1 ppm, 5 ppm, 10 ppm, and 25 ppm (for the others, as Cu and Mo) obtained by dilution from
a MERCK stock standard solution for each element, containing 1000 mg•L-1 of Cu, Mo, and Se.
Statistical analysis - Statistical analysis was performed using the software of VassarStats:
Website for Statistical Computation (http://vassarstats.net/). One-Way ANOVA was performed for
all samples' mineral concentrations, and when ANOVA generated p≤0.05, means comparison was
carried out by all-pair Tukey HSD Test.
327
Results and discussions
The mean Cu, Mo, and Se contents of hair samples from clinically healthy pet cats are
presented in Table 1 and expressed as mg•kg−1.
In this study, the effect of living conditions on Cu, Mo, and Se concentrations in the pet
cat hair was observed, but habitat influenced statistically insignificant hair Cu, Mo, and Se levels
in pet cats. The highest mean value of Cu was registered in the group of cats above 5 years kept
indoors (26.736 mg•kg−1), significantly different compared to hair Cu mean level of cats below 5
years kept in the same living conditions. Considering the animal gender, the mean content of Cu
in males was higher in indoor pet cats hair (19.473 mg•kg−1), and in females it was higher in
outdoor pet cats hair (14.32 mg•kg−1). Indifferent of gender, hair Cu mean levels were higher in
pet cats above 5 years kept indoor (26.736 mg•kg−1), and in outdoor pet cats below 5 years (15.064 mg•kg−1). Also, it was observed that hair Cu mean levels in pet cats below 5 years registered levels
significantly different reported to animals’ living conditions. In this study, it was observed no
significant differences between Cu mean hair levels in investigated groups of pet cats kept indoors
or outdoors, independent of gender or age.
Table 1. Cu, Mo, and Se mean levels in pet cats hair samples (mg•kg−1)
Element no Cu Mo Se
Males in 5 19.473 a 0.215 a 2.111 a
out 5 10.330 a 0.358 a 2.279 a
Females in 5 10.584 a 0.084 a 2.317 a
out 5 14.320 a 0.160 a 2.463 a
Age
below
5
in 7 10.011*a 0.087 a 2.310 a
out 3 15.064* a 0.222 a 2.257 a
above
5
in 3 26.736 b 0.296 a 1.991 a
out 7 11.288 a 0.274 a 2.420 a
Habitat in 10 15.028 0.149 2.214
out 10 12.547 0.259 2.371
All animals 20 13.853 0.204 2.293
* Significant differences at p ≤ 0.05. The comparison can be made only between habitats for the concentration of one
element and not between different elements concentrations. a,b Levels not connected by the same letter are significantly different (p ≤ 0.05). The comparison can be made only
between sex or age in the same habitat for the concentration of one element and not between different elements
concentrations.
Hair Mo levels registered reversed values reported to Cu – higher levels when Cu registered
lower values and lower concentrations when Cu was higher. Mo registered highest mean level in
outdoors males (0.358 mg•kg−1), and lowest mean level in indoors females (0.084 mg•kg−1).
Reported to age, Mo mean hair levels registered higher levels in above 5 years pet cats indifferent
of living conditions, and higher mean levels in outdoors pet cats below 5 years and in indoors pet
cats above 5 years. Insignificant differences were observed between Mo mean hair levels in pet
cats kept indoors or outdoors, indifferent of gender or age.
328
The highest mean value of Se was registered in the group of female cats kept outdoors
(2.463 mg•kg−1), insignificantly different compared to hair Se mean level of indoors female cats and
also males kept outdoors. The mean Se hair levels registered insignificant differences for the same
habitat in pet cats below 5 years and above 5 years. Independent of habitat, hair Se mean levels
were higher in females compared to males. In this study, hair Se registered higher mean levels in
outdoors pet cats, independent of habitat, gender, and age, excepting pet cats below 5 years were
the values registered were reversed. The lowest mean Se hair level was registered in the group of
pet cats above 5 years kept indoors (1.991 mg•kg−1).
In the available literature, there are no data concerning Mo content in the hair of domestic
cats. The obtained results could only be compared with the data concerning Cu and Se in control
groups of pet cats. While analyzing mineral and heavy metal content in the hair of cats in
relationship with kidney failure, Badea et al. (2016a) obtained for clinically healthy animals
(control group) the results for Cu of 0.94 mg•kg−1 in females and 0.92 mg•kg−1 in males, and also,
hair Se ranged from 0.07 mg•kg−1 in males, and 0.15 mg•kg−1 in females, which are in both trace
minerals, much lower compared to those registered in our study for the same groups, independent
of habitat. The same pattern was observed reported to age in both trace minerals, which are also
much lower compared to those registered in our study for the groups below 5 years and above 5
years, independent of habitat.
Cu and Se content in the hair of female pet cats from the group of healthy animals, which
represented control group in a study on hair mineral content analysis in cats with different liver
disorders amounted to 0.78 mg•kg−1 for Cu and 0.103 mg•kg−1 for Se in females above 8 years
(Goran et al. 2017). These values are much lower in the case of Cu and approximately in the same
range, but lower for Se than those obtained in our study.
Conclusions
Even in this study, Cu, Mo, and Se levels in pet cats hair were not significantly influenced
by the living conditions, hair may be considered as an indicator for the mineral status of cats in an
urban area.
Mean value of hair Cu in group of cats above 5 years kept indoors was significantly
different compared to hair Cu mean level of cats below 5 years kept in the same living conditions.
Hair Cu in younger pet cats registered significantly different mean levels dependent of
habitat.
Mo registered lower mean levels when mean Cu levels were higher and higher levels when
Cu was lower, indifferent of habitat, sex, and age.
Hair Se registered higher mean levels in outdoors pet cats, independent of habitat, gender,
and age.
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Copper-releasing, borate-based glasses with antibacterial properties:
synthesis and in vitro characterization
Cristina LELESCU1*, Aurel MUSTE1, Marian TAULESCU1, Gheorghe BORODI2,
Marin ȘENILĂ3, Lucian BARBU-TUDORAN2, Răzvan ȘTEFAN1
1Faculty of Veterinary Medicine, University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca
2National Institute for Research and Development of Isotopic and Molecular Technologies, Cluj-Napoca,
3Reseach Institute for Analytical Instrumentation, Cluj-Napoca *[emailprotected]
Abstract
In this study, glasses within the system (60-x) B2O3x ZnO34CaO1CuO, with x=5, 10, 15, 20, 25
mol% and with B2O3/ZnO ratios 11; 5; 3; 2; 1.4 have been synthesized and characterized in vitro. After
being immersed in simulated body fluid (SBF) and saline solution, weight loss reduction and pH
measurments, followed by inductively coupled plasma optical emission spectometry (ICP-OES), scanning
electron microscopy (SEM) and X-ray diffraction (XRD) analysis were performed, in order to evaluate the
changes in glass morphology. In vitro biodegradation and surface reaction were observed in all of the
glasses, especially in the x=10, 15, 20 samples. SEM and XRD results revealed the presence of a
hydrotalcite-like structure (double layered hydroxid) at the aqueous solution-glass surface interface, while
Cu, Zn, Ca and B ions, with proangiogenic properties, were detected in the immersion fluid.
Keywords: copper, borate glasses, in vitro, biological ions;
Introduction
Bioactive glasses are a category of vitreous biomaterials with remarkable bioactivity and
biocompatibility, which makes them highly promising in the medical applications field. In recent
years, there has been a significantly growing interest in the use of borate-based bioactive glasses
due to their excellent properties in bone regeneration and beyond. [4,24]. Low chemical durability,
faster and more complete degradation in comparison with silicate glasses, and a controllable
conversion to hydroxyapatite (HA) are among the well known characteristics of boron-containing
glasses [10]. Still, an extensive and ongoing process of research is required to provide prospects
for their use in soft tissue engineering [20].
A controllable degradation rate of the bioactive glass is desirable, taking into account that
it should be similar to the rate of new tissue formation, and a high chemical durability results in a
longer and less complete degradation rate upon immersion in aqueous solutions. [10,18,20]. This
process is accompanied by dissolution of ionic compounds in the fluid in which the bioactive glass
was immersed, resulting in pH and ionic concentration changes of the aqueous solution over time
[6]. Also, to form a strong bond with the surrounding tissue, the glass surface must undergo a
specific conversion phenomenon when immersed in simulated body fluid (SBF), leading to the
fomation of a hydroxiapatite (HA)-like surface layer [8]. Therefore, in vitro characterization of
these processes is essential to predict the in vivo behavior of the materials, and their applicability
in tissue engineering [20].
Weight loss measurements and pH monitoring of the aqueous immersion solution are
simple and relevant methods for assessing glass degradation rate, as they accompany these
processes by changes in ion concentration of the surrounding aqueous medium [10]. Scanning
electron microscopy (SEM) coupled with energy-dispersive X-ray analysis (EDS) is used to
measure microstructural surface changes after immersion [16], while newly formed crystalline
331
phases can be detected by X-ray diffraction (XRD) analysis [10]. Concentration of ions released
into deionized water following immersion, can be accurately measured by using inductively
coupled plasma optical emission spectometry (ICP-OES), being an essential tool for predicting
effects of the biomaterial on the surrounding tissue, including: cell growth stimulation, growth-
factor enhancement [22], angiogenesis promotion [15] and gene expression regulation [23].
The aim of this paper was to thoroughly characterize 5 compositions of bioactive glasses
derived from the B2O3ZnOCaOCuO system, by measuring the weight loss and pH changes of
the glass, after being immersed in aqueous solutions at 37°C. Additional SEM-EDS and XRD
analysis were performed to characterize microstructural surface changes, while ICP-OES was
completed after immersion of the glass in deionized water at 37°C. The results are intended to
anticipate the biological behavior of the glasses, and their applicability in tissue engineering.
Materials and methods
Glass synthesis
Glasses within the system (60-x) B2O3x ZnO34CaO1CuO, with x=5, 10, 15, 20, 25 ZnO
mol% and with B2O3/ZnO ratios 11; 5; 3; 2; 1.4 were prepared with high purity raw reagent-grade
chemicals. In order to obtain five different compositions of borate-based bioactive glasses, zinc
oxide (ZnO), copper oxide (CuO), boric acid (H3BO3) and calcium carbonate (CaCO3) powders
were weighed, mixed and hom*ogenized before being melted at 1230°C for 25 minutes in sintered
corundum crucibles. The compositions were rapidly cooled at room temperature by quenching the
molten glasses; subsequently, the samples were crashed in an agate mortar and passed through a
sieve in order to finally obtain particles with a diameter (d) < 0.075 mm.
Degradation behavior and pH assessment of the borate-based glass samples in 0.9%
saline solution
In order to evaluate the degradation behavior of the bioactive glasses, similarly sized
samples were weighed before being soaked in 5 ml of normal saline solution (0.9%) and incubated
at 37°C for 7 days. The weight reduction was assessed by measuring the relative weight loss (Δm)
in all samples, using the following equation: m= 100(mi-mf)/mi, where mi is the initial mass,
found in the samples before immersion and mf is the measured weight after 7 days of soaking in
saline solution. Each sample was removed from the solution and wiped gently with filter paper to
eliminate the fluid from the surface before being weighed. Consequently, the glass surface was
microscopically evaluated at the end of immersion time (Olympus BX51).
Ionic dissolution assessment 100 mg of glass powder from each sample were soaked in 5 ml of deionized water, being
subsequently incubated at 37°C for 5 days. The concentration of ions (copper, zinc, borate, calcium
and aluminium) released during the degradation process were measured by ICP-OES analysis
(FMD-07, Spectro Analytical, Germany). Multielement calibration solutions prepared from a stock
solution of 1000 mg L-1 (Merck, Darmstadt, Germany) were used to calibrate the instrument. The
estimated error in the measurement rate was +/- 5%.
Evaluation of the surface microstructural and chemical composition changes
Changes in the chemical and mirostructural properties of the samples, resulting from the
interaction with aqueous solutions, were assessed after immersing the glasses in normal saline
solution 0,9% for 7 days, at 37°C. The surface texture and morphological evaluation was performed
by SEM, while using XRD analysis (Bruker D8 Advance), the crystalline phases were quantificated
following interaction with the aqueous medium.
332
Results and discussions
Five glass compositions belonging to the (60-x) B2O3x ZnO34CaO1CuO system, with
x=5, 10, 15, 20, 25 ZnO mol% and with B2O3/ZnO ratios 11; 5; 3; 2; 1.4 were obtained by melting
technique (fig. 1). Samples were rapidly cooled at room temperature, being subsequently crushed
and passed through a sieve, resulting in particles with a diameter of <0.075 mm (fig. 2).
Fig. 1 Macroscopic appearance of the (60-x)
B2O3x ZnO34CaO1CuO with x=10 sample,
after being quenched and crushed
Fig. 2 Macroscopic appearance of the (60-x)
B2O3x ZnO34CaO1CuO with x=5 sample, after
being crushed and sieved to <0.075 mm particles
In the present study, granular forms of glass particles were obtained in order to increase
the glass surface area, thereby increasing the reactivity of the sample surface in the surrounding
physiological fluid.
Macroscopic aspects of the samples following immersion After 7 days of immersion in saline solution at 37°C, a newly formed layer of
approximately 0.5 mm thickness, very brittle, whitish and easily ruptured was noted at the surface
of the glasses (fig. 3), especially in the (60-x)·B2O3·xZnO·34CaO·1CuO, where x= 10; 15; 20
samples. Microscopically, the newly formed layer exhibited a crystallized appearance, while the
underlying surface layer presented a smooth aspect, lacking pre-existing discontinuities (fig. 4).
Clusters formed via agglomeration of particles were significantly decreased in number and size,
whilst in some regions, due to the thickness of the newly formed layer and its opacity, the
microscopic evaluation could not be achieved.
Fig. 3 Macroscopic appearance of the newly
formed glass surface layer after 7 days of
immersion in normal saline 0,9% in the (60-x)
B2O3x ZnO34CaO1CuO with x=10 sample
Fig. 4 Microscopic appearance of the surface layer
after 7 days of immersion in normal saline 0,9%
of the (60-x) B2O3x ZnO34CaO1CuO with x=20
sample (100x)
333
A remarkable, dynamic process has been noted at the aqueous solution-glass surface
interface on microscopic evaluation of the samples, consisting of an uninterrupted deposition of a
thin layer, with a crystallized appearance (fig. 5-6).
Fig. 5-6: Continuous deposition of a thin layer on the sample surfaces, after 7 days of immersion in
SBF, at 37°C
Degradation behaviour and pH assessment of the bioactive glass samples
The average weight reduction (Δm) for all of the five samples after immersion in 0.9%
saline solution, was found to be 0.86%. The highest weight reduction was recorded in the (60-
x)·B2O3·xZnO·34CaO·1CuO, where x=10 sample (Δm= 1.76%), while the lowest weight
reduction was identified in the (60-x)·B2O3·xZnO·34CaO·1CuO, where x=25 sample (Δm=
0.03%). Although weight losses are relatively low, they indicate the ocurrance of a degradation
process; however, a reduced degradation and conversion rate is rather characteristic for silicate
glasses, and represents a drawback by hindering the coordination between the bioactive glass
degradation rate and tissue regeneration rate [7,20].
The starting pH value of the 0.9% saline solution at 37°C was 5.4. After 7 days of
immersion, the most significant increase in the pH value was recorded in the (60-
x)·B2O3·xZnO·34CaO·1CuO, where x=15 sample, with 3.5 units. The saline solution has
undergone the smallest change in pH in the (60-x)·B2O3·xZnO·34CaO·1CuO, where x=25 sample,
with 1.9 units. Changes in the pH take place due to changes in ionic concentrations, as a result of
the degradation process [10,20]. A previous study has shown a correlation between increasing the
B2O3 content in the sample and increasing pH of the immersion solution [7]. Huang [10]
highlighted that, by increasing B2O3 content, a very rapid pH increase of the aqueous solution was
caused, reaching a limit value in 50 hours; however, the final pH value was higher in the samples
with a decreased B2O3 content, where the limit value was reached after 500 hours. In the present
study, pH evaluation was performed after 168 hours of immersion and showed a lower pH increase
in the sample with the highest B2O3 content. Increasing B2O3 content results in a lower chemical
durability, which leads to a faster reaction with the aqueous medium, and therefore to the rapid
change of the pH, but it’s limiting value is detemined by the ionic concentrations released in the
solution, as well as by their acidity and basicity [10].
Ionic dissolution products
In order to measure the ions concentrations released into the deionized water, the ICP-OES
analysis was performed; results in all of the five samples are presented in Fig. 7.
334
Sample Cu (mg/L) Zn (mg/L) B (mg/L) Ca (mg/L) Al (mg/L)
x=5 0.038 0.048 530 770 3.6
x=10 0.054 0.041 506 730 3.6
x=15 0.075 0.044 544 840 2.7
x=20 0.045 0.049 510 810 1.1
x=25 0.066 0.054 650 1270 1.2
Fig. 7: Ion concentrations of deionized water following immersion of the glass samples
The highest concentrations of B, Zn and Ca were found in the (60-
x)·B2O3·xZnO·34CaO·1CuO, where x=25 sample (650 mg/L, 0.054 mg/L and 1270 mg/L
respectively). Cu reached a maximum level in the (60-x)·B2O3·xZnO·34CaO·1CuO, where x=15
(0.075 mg/L) sample, while Al levels were found in all of the five samples, with the highest
amounts being detected in the (60-x)·B2O3·xZnO·34CaO·1CuO, where x=5, 10 samples (3.6
mg/L). The presence of trace amounts of aluminium is explained by the high temperature and the
long melting time during sample synthesis, which caused contamination from the walls of the
crucible.
The biological significance of Cu is given by its anti-inflammatory, anti-infectious,
antibacterial and proangiogenic properties [1]. A remarkable cellular distribution of Cu ions has
been revealed in human endothelial cells, when induced to undergo angiogenesis [5]. Zn ions are
required in various enzymatic activities and anti-inflammatory processes, possess a remarkable
antimicrobial activity and are strongly involved in protein synthesis [2,12]. They appear to be
actively involved in collagen synthesis and play a role in cell membrane stabilization, intracellular
signaling and wound healing [14]. Increased levels of Zn were identified at the wound margins
within the first 24 hours, while even higher levels were detected during epidermal granulation and
proliferation [9,13]. Boron ions are apparently involved in the synthesis of collagen and
proteoglycans, increase the turnover of extracellular matrix, and also promote protein
phosphorylation [19]. Al has certain astringent, antacid and antibacterial properties, its toxic effects
being dose dependent [17]. Microstructural characterization
SEM photomicrographs of the (60-x)·B2O3·xZnO·34CaO·1CuO, where x=10, 15 samples
are shown in fig. 10. Cauliflower-shaped agglomerates (fig. 8A), sphere clusters (fig. 8B), and also
well defined, regular geometric shapes (fig. 8C-D) were observed at the surface of the outermost
layer.
335
Fig. 8 SEM images of the (60-x)·B2O3·xZnO·34CaO·1CuO, where x=10, 15 samples.
A : x=10, scale:50 µm; B: x=10; scale:50 µm; C: x=15, scale:500 µm;
D: x= 15, scale:100 µm
Following imersion in saline solution, XRD patterns of the samples were relatively
similar (fig. 12), showing peaks corresponding to zinc aluminium carbonate hydroxide hydrate
[Zn6Al2(OH)16CO3.4H2O], a hydrotalcite-like structure, also known as double layered hydroxid
(strongest line at 2θ = 11.65°). The importance of this compound lies primarily in its catalytic
behavior, particularly being a substrate for the efficient immobilization of biological materials
[3,21]. The development of this interface layer is a result of the interaction between Zn and Al ions,
under alkaline hydrothermal conditions [11]. In his study, Koh [11] developed this chemical
compound, and successfully used it as a synthetic substrate for hexagonal-patterned ZnO nanorods.
A B
C D
[
T
y
p
e
a
q
u
o
t
e
f
r
o
m
t
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e
d
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c
u
m
e
n
336
Fig. 12: X-ray diffraction patterns of the (60-x) B2O3x ZnO34CaO1CuO system,
with x=10, 15, 20 after immersion in SBF
Aluminum sulphate hydroxide hydrate is another compund detected by XRD analysis at
the sample surfaces, the most intense peak being centered at 2θ = 8.31˚. The XRD scan also
contained diffraction peaks corresponding to NaCl, originating form the immersion fluid.
Diffraction peaks could only be detected and assigened in the x=10, 15 and 20 samples.
Conclusions
Five different borate containg glasses with a composition in the (60-
x)·B2O3·xZnO·34CaO·1CuO system, were synthesized and characterized by various methods,
including ICP-OES, SEM-EDS, and XRD, in order to predict their applicability in tissue
engineering. In vitro weight loss and pH measurements showed a lightly degradable behavior in
all of the samples, while surface reactivity of the glasses was studied microscopically. None of
these properties appeared to be linearly dependent on the boron concentration of the glass samples.
SEM micrographs captured at the surface of the samples revealed the presence of various distinct,
well-defined, regularly shaped structures, especially in the (60-x)·B2O3·xZnO·34CaO·1CuO,
where x = 10; 15 samples. The XRD patterns of the samples showed the existance of a newly
formed, hydrotalcite-like structure at the aqueous solution-glass surface interface, with remarkable
biological properties. These results indicate a highly promising stimulatory potential of these
glasses, by releasing various ions with multiple activities, related to their proangiogenic and
antibacterial properties.
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338
Searches on the application of a method of induction and
syncronization of estrus in cows postpartum based on two doses of
GnRH and prostaglandin F2 α, with programmed insemination
Elena RUGINOSU, S.I BORȘ, Ș. CREANGĂ, D.L. DASCĂLU, Mădălina Alexandra DAVIDESCU
Stațiunea de Cercetare Dezvoltare pentru Creșterea Bovinelor Dancu Loc. Dancu, jd. Iași
[emailprotected]
Abstract
The researches were realized in the dairy cattle farm from S.C.D.C.B. Dancu, Iasi, on an
experimental lot, composed of 41 cows of Romanian Black Spotted breed (BNR), (Lot E), compared to a
control group composed of 45 cows (Lot M). The purpose of this study was to determine the effectiveness of
using a new protocol for the induction and synchronization of estrus at dairy cows, using the hormonal
products, such as GnRH and PGF2α. The treatments applied to the cows from the experimental group were
performed between 31.01.2017 and 23.06.2017, within an average value of 52 days after calving. The
therapeutic protocol consisted in the following: administration of the first dose of GnRH (2.5 ml,
Receptal=10 μg Buserelin, intramuscularly) on day 0, (regardless of the stage of the estrous cycle);
administration of one dose PGF2α (2ml Estrumate=500 mcg Cloprostenol) on the seventh day;
administration of the second dose of GnRH on the ninth day and artificial insemination at 18-24 hours after
the second dose of GnRH, (without estrus detection). The application of this therapeutic protocol for estrus
induction and synchronization at dairy cows with two doses of GnRH and PGF2α in the 45-80 days
postpartum period has reduction effects of the intervals: calving to the first insemination and conception,
with a positive impact on the reproductive management of dairy farms and the economic implications by the
reduction of unproductive costs and of the workforce for the detection of cows estrus.
Key words: dairy cows, estrus induction and synchronization, GnRH, prostaglandin F2α, fertility
Introduction
During the last period was found a decline in reproductive efficiency, particularly in highly
productive cows, due to the poor detection of the estrus, caused by the reduced intensity of heat
signs, the inadequate time of observation of the animals in large farms and the deterioration of
climatic conditions. Most studies indicate an association between lower fertility of cows with high
milk yields and decreases concentrations in steroid hormone (estrogen-E2 and progesterone-P4),
(1, 2, 9, 15, 16, 17).
Research in the field shows that treatments for cow estrus induction and synchronization
may have positive effects on reproductive activity, especially in animals maintained in the free
system, facilitating estrous detection and increasing the conception rate by inseminating the
animals at the optimum time in relation to ovulation. Of the different protocols for estrus induction
and synchronization in dairy cows and meat, the protocol named Ovsynch, which uses a
combination of injections of GnRH and PGF2α, is widely used because eliminates the estrus
detection and allow fixed time insemination programmed at ovulation optimum time, resulting in
normal fertility after insemination at the end of treatments (1,2,3,4,5,7,9,10,11,15,16,17).
Various studies have shown that Ovsynch is an extremely economical and efficient
strategy to improve reproductive performance of high performance dairy cows (3,12,13,14).
During a cow's sexual cycle, there are 2-3 waves of ovarian follicular growth, each
follicular wave having an average life of 7-10 days, progressing through different stages of
development, from appearance, selection, dominance, atresia or ovulation. A dominant follicle,
mailto:elena.ruginosu54@
339
capable of ovulation is present only at certain times during each wave. Given the dynamic growth
of ovarian follicles in a sexual cycle, the Ovsynch therapeutic protocol was developed, which
aiming to achieve the following effects: reprogramming follicular waves by administering the first
dose of GnRH, the luteolysis of a probable luteal body by administering of PGF2α to 7 days and
determining the dominant follicle ovulation expected to be present after PGF2α by administering
the second injection of GnRH at 48-56 hours later, thus facilitating fixed-time insemination,
without resorting to detection of heat (1,2,6,7,9,15).
Major implications of progesterone (P4) concentration were reported at the time of the first
dose of GnRH on the induced LH wave. It was established that in order to have a successful
Ovsynch protocol, progesterone (P4) concentration at first GnRH1 treatment should usually be
above 1 ng / ml), while in the second GnRH2 treatment, the (P4) concentration to be less than 0.5
ng / ml (5.16).
Some authors report that conception rates in lactating cows that benefited from the
Ovsynch protocol were similar to those of cows that were inseminated to spontaneous postpartum
estrus (Pursley et al., 1997a, b).
Other recent reports have indicated that although the use of these estrus therapeutic
protocols in cows has many advantages, which would come from an effective regulation of sexual
cycle and facilitating reproductive management in farms with large livestock, however, the
methods of control of sexual cycle have variable results, some being contradictory and questionable
(3,4,5,9,10,11,12,13,14,15,16,17).
Variability of Ovsynch protocol results consist in the proportion of non-cycling postpartum
cows, the follicular dynamics of each cow in the herd or the ability of farm personnel to properly
implement this protocol (7,15,16).
The purpose of present researches was to establish the efficacy of estrus induction and
synchronization protocol based on the combination of injections of GnRH and PGF2α, taking into
account the positive effects on reproductive management of dairy cows maintained in the free
system, but also the variability of treatment outcomes, reported by various authors.
Material and methods
Research has been carried out on zootechnical base from Research and Development
Station for Breeding Cattle (S.C.D.C.B.) Dancu, Iasi, on an experimental lot (Lot E), consisting of
41 cows compared to a control group of 45 cows (Lot C) in Romanian Black Spotted breed, freely
maintained in a modernized shelter with a capacity of 200 heads, (Figure 1).
Figure 1. Maintenance system of the cows in the experimental group
340
The treatments for estrus induction and synchronization of cows from the experimental
group (E lot) were performed between 31.01.2017 and 23.06.2017, the average interval from
calving to treatment being 52 days (with variations between 42-70 days).
The therapeutic protocol consisted in the administration at cycling or non-cycling
postpartum cows, regardless of ovarian formations (follicles or luteal body) two doses of 2.5 ml
(10 μg) intramuscularly of GnRH (Receptal), separated by a dose of 2 ml prostaglandin F2α
(Estrumate) intramuscularly (containing Cloprostenol 250 μg / ml, Intervet International BV,
Netherlands) at 7 days after the first administration of GnRH (Receptal, 2 ml, intramuscularly).
Two days after the injection of prostaglandin F2α, a second dose of GnRH (Receptal, 2 ml,
intramuscularly) was given. Considering the moment of ovulation, the second injection of GnRH
was given in the afternoon between 14-15 hours for the insemination to be carried out in the next
morning between 8-9 hours, thus achieving the range of 18-19 hours from the second injection of
GnRH, which coincides with the production of ovulation. At 18-24 hours after the second dose of
GnRH, (Receptal, 2.5 ml) artificial insemination of cows was performed, without resorting to
detection of heat (Figure 1).
GnRH PGF2α GnRH fixed time IA
(Receptal, 2,5 ml) (Estrumate, 2 ml) (Receptal, 2,5 ml)
0 1 2 3 4 5 6 7 8 9 10 days
7 days 2 days 18-24 hours
Figure 2. The protocol for induction and synchronization of estrus in cows
based on 2 doses GnRH and PGF2α
The effectiveness of treatments applied to cows in the experimental group was determined
by determining the intervals from treatment to conception, calving to conception, gestation index
and conception rate on total insemination, as well as after the first, second, third and third
insemination.
Results and discussions
The artificial insemination of cows took place after the second dose of GnRH (with or
without estrus signs) in 97,56% of cases (40 of 41 cows), of which: 87,81 % (36 of 41 Cows)
according to the protocol at fixed time (18-24 hours) and 9,75% (4 of 41 cows) which showed
estrus were early artificially inseminated (with second administration of GnRH. One cow (2,43%)
was not inseminated due to the presence of a genital inflammatory process.
The treatment-conception interval recorded an average of 32.8 ± 10.6 days (with
variations between 1-110 days), (Table 1).
341
Table 1. The artificial insemination of cows in experimental group
after the estrus induction and synchronization treatments
with 2 doses GnRH and prostaglandin F2α
The comparative analysis of cow’s reproductive activity revealed that the average value
for the first postpartum insemination interval was with 29 days lower in the experimental group
(62 ± 1.05 days) compared to the control group (90.94 ± 8.00 days). However, there were no
differences in the average conception interval , the values being close to the two batches close in
the two groups (94.93 ± 10.4 days, in the experimental group and 93.72 ± 9.86 days, respectively,
in control group), (Table 2).
Table 2. The reproductive activity of cows in the experimental group (n = 41 cows),
after the treatments for estrus induction and synchronization treatments
compared to control group without treatments (45 cows)
-------------------------------------------------------------------------------------------------------------------
cows /lot E artificially inseminated cows (IA) after treatments, of which:
total IA IA at 18-24 hours IA early without IA
after in day of
second GnRH second GnRH
fixed time
----------------------------------------------------------------------------------------------------------------
-
n n % n % n % n %
------------------------------- --------------------------------------------------------------------------------
-
41 40 97,56 36 87,81 4 9,75 1 2,44
----------------------------------------------------------------------------------------------------------------
-
---------------------------------------------------------------------------------------------------------------------------
----
Intervals, (days)
---------------------------------------------------------------------------------------------------------------------------
-
specification calving- calving- treatments- calving-
values treatments the first conception conception
insemination
postpartum
lot E lot E lot C lot E lot E lot C
---------- ---------------------------------------------------------------------------------------------------------------
---
average 52±1,03 62±1,05 90,94±8,00 32,8±10,6 94,93± 10,4 93,72±9,86
minim 42 52 35 1 53 35
maxim 70 80 242 110 180 174
--------------------------------------------------------------------------------------------------------------------------
---
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The analysis of the conception rate after estrus induction and synchronization treatments
reveals on total insemination and after the first two inseminations with 6,39% and 8,4% higher
values in the experimental group compared to control group (37, 50% - lot E vs. 31,11% - lot C
and 35% - lot E, vs. 26,6% - lot C), (table 3).
Table 3. The conception rate of cows in the experimental group
after treatments for estrus induction and synchronization treatments
compared to control group without treatments
By applying this therapeutic protocol to cows for induction and synchronization of estrus,
it was found that in 9,75% of cases showed estrus and were artificially inseminated early (in the
days of the second administration of GnRH) and in 87,81% of cows did not show estrus, the
artificial insemination being performed in fixed time within 18-19 hours after the second
administration of GnRH.
This finding is confirmed by other authors who show that between 80 and 90% of the cows
treated with this protocol are not observed in the estrus, GnRH injection causing the ovulation
before the cow displays signs of estrus, approximately 26-32 hours after the second injection of
GnRH (8,10).
Following the studies by Fricke et al., 1998, the ovulation of one dominant follicle, in
response to the second GnRH injection occurs in about 85% of highly productive milk cows who
receive this protocol.
Some authors have achieved the highest conception rate of cows that have been artificially
inseminated within 8-24 hours after the second injection of GnRH, indicating a considerable
flexibility in insemination time after the second injection of GnRH, the conception rates being
lower only when cows were inseminated after ovulation time.
Analysis of the ovarian profile at the initiation of the estrus synchronization protocol in
relation to the gestation status of the cows has shown that the conception rate after treatment
depends on the trophic state of the ovaries, the phase of the estrous cycle in which it is applied and
the ovarian follicles dimensions Table 4).
The influence of estrous cycle phase in cows at the time of initiation of treatments on the
conception rate was also indicated by other authors, who report that the ovulation synchronization
rate is higher when Ovsynch is initiated in the middle of the estrous cycle. When the Ovsynch
--------------------------------------------------------------------------------------------------------------------------
Cows Pregnant cows, of which:
inseminated
after Total at I-a IA at I –a + a-II-a IA
treatments
Lot E Lot E Lot C Lot E Lot C Lot E Lot C
n % n % n % n % n % n % n %
---------------------------------------------------------------------------------------------------------------------------
----
40 97,57 15 37,5 14 31,11 7 17,5 7 15,5 14 35,0 12 26,6
------------------------------------------------------------------------------------------------------------------------
----
343
protocol began on day 5 through day 9 of the estrous cycle, there were several cows (> 90%) that
ovulated after the first dose of GnRH. Beginning in the middle of the cycle, Ovsynch led to smaller
ovulatory follicles and higher gestation rates. Synchronization rate was 91% when Ovsynch was
started on day 1 to 12 of the estrous cycle and 80% when Ovsynch was started on day 1 -22 (17).
Table 4. Ovarian profile at the initiation of the estrus synchronization therapeutic protocol
with 2 doses of GnRH + prostaglandin F2α in relation to gestational status after treatments
Ovarian formations Pregnant cows,
at the initiation of at total inseminations, of which: at the first
insemination
therapeutic protocol
for cows estrus synchronization
1-2 large sized follicles (12-18 mm
1-2 medium sized follicles (8-11 mm) 1 1
1 large sized follicles (15 mm) 1 -
1 Luteal body
2 medium sized follicles (8-11 mm) 2 -
1 Luteal body
1-2 medium sized follicles (8-11 mm) 1 1
1 Ovarian hypotrophy
1-2 medium sized follicle (8-11 mm)
on both ovaries 2 -
2-4 small sized follicles (4-6 mm) 3 1
on both ovaries
2 small sized follicles (4-6 mm) 3 1
1 Ovarian hipotrophy
1 large sized follicles (15 mm) 1 1
1 medium sized follicles (10 mm)
2-3 small sized follicles (4-6 mm)
TOTAL pregnant cows 15 7
The research presented in this paper highlights that this therapeutic protocol based on the
combination of injections of GnRH and PGF2α for the induction and synchronization of estrus in
cows within the 42-70 days postpartum period has effects of decreasing the average interval
between calving and the first postpartum insemination and higher conception rates, compared to
the control group.
Although this protocol requires cows to be handled 3-4 times, heat detection are minimized
or eliminated, making cow's artificial insemination more practical and economically feasible for
dairy cows and increasing the number of cows that are inseminated in the first 60 days after calving.
Conclusions
- Cows who benefited in postpartum by estrus synchronization treatments (experimental
group) were artificially inseminated after the second dose of GnRH (with or without signs of
344
estrus) in 97,56% of cases, of which: 87,81% in according to the protocol at fixed time (18-24
hours) and 9,75% who showed estrus and were artificially inseminated early, (in day of GnRH
second administration).
-The average calving -treatment interval in the experimental group was about 52 ± 1,03
days (with variations between 42-70 days).
- The treatment-conception interval in the experimental group had an average value of 32,8
± 10,6 days (with variations between 1-110 days).
- The conception rate in the experimental group on total inseminations had higher values
with 6,39%, and after the first two inseminations with 8,4% compared to the cows in the control
group (37,50% - lot E vs. 31,11% - lot C and respectively 35% - lot E vs. 26,6% - lot C).
- By the therapeutic protocol based on the combination of injections of GnRH and
prostaglandin F2α for the induction and synchronization of estrus in cows the minimization or
elimination of heat detection is made, making artificial insemination of cows more practical and
economically feasible for dairy cattle farms and increasing the number of cows which are
inseminated within the first 60 days after calving.
This work was conducted under the Sectorial Plan for Research and Development of
Ministry of Agriculture and Rural Development for the years 2015-2018 "Agriculture and Rural
Development - 2020 ADER", contract number 5.3.2/03.11.2015
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346
vagin*l smear, progesterone levels, and ultrasound examination of the
ovaries as methods of determining the moment of ovulation in bitches
comparative study
G. OTAVĂ, C. MIRCU, Violeta IGNA, Simona MARC ZARCULA, D. LO PRESTI Faculty of Veterinary Medicine, University of Agricultural Sciences and Veterinary Medicine of Banat ‟ King Michael I of Romania” (USAMVB), Timisoara – 300645, Calea Aradului, nr. 119,
[emailprotected]
Abstract
Researches in the scientific literature reveal that the study of vagin*l citology and interpretation
of progesterone values do not represent certain methods to determine the ovulation time. 6 different breed
have been investigated in this study (Labrador Retriever, Tibetan Mastiff, Bichon Maltese, West Highlander
White Terrier and the Bucovina Shepherd). All es were monitored in terms of cyto-vagin*l smear, the P4
level and ultrasound examination to determine the ovulation moment. The rapid disappearance of the
follicular antrum cavity, correspondent to ovulation, was detected only with two. Although ultrasound
changes during the estrous cycle were well-studied, the exact ovulation moment cannot be predicted
accurately. To optimize the results of determining the ovulation moment it is recommended to collate the
ultrasound examination with at least one of the other two methods of investigation.
Key words: ovulation time, bitch , ultrasound
Introduction
Bitches are mono-estrous animals with a single reproductive cycle. Because ovulation
occurs once or twice a year and due to the very high variaty among individuals within the same
breed and among breeds, it is very difficult to set a very accurate method for ovulation timing.
Imagining and developing a non-invasive method of monitoring follicular development and
ovulation is necessary both for research and for clinical practice. Development of some high-
performance ultrasound apparata has led to substantial progress in this area lately.
Until now, the ovulation moment has been set by collating the two methods of medical
investigation- determinating the progesterone level and studying the vagin*l cytology . The study
of literature concerning the determination of the ovulation moment by determining the
progesterone level reveals vastly different data. Thus, progesterone values ranging from 4 to 10
ng/ml are set through clinical studies. In this study we wanted to obtain information about the
effectiveness and coordination of the three methods of determining the ovulation moment.
Materials and methods
Six different breeds have been monitored in this study (Labrador Retriever, Tibetan
Mastiff, Bichon Maltese, West Highlander White Terrier and the Bucovina Shepherd). Taking into
account the very large differences among individuals of the same breed and among dog
breeds,regarding the ovulation moment opposite the sexual cycle, pet owners were advised to
bring the females for monitoring the estrous phase of the sexual cycle when the blood leak
diminished , both chromatically and quantitatively. Thus, the owners were not called according to
a fixed interval from the sexual cycle onset.
An attempt was made to investigate the vagin*l cytology, the progesterone levels and
ultrasound appearance of the ovaries in the females. To perform the cytovagin*l smear, I needed a
rod of wood to which a small amount of cotton wool was attached by rotating the rod. This
improvised item was introduced into the vagin*l vestibule on a nearly vertical direction from the
bottom up, after a previous distance of vulvar lips performed by the same vet tehnician with the
mailto:[emailprotected]
347
opposite hand. After penetration into the vagin*l vestibule the rod is placed in horizontal position
in order to set its tip into the anterior vagin*.
According to the literature there are differences concerning the interpretation based on
cytovagin*l smear in es between the vagin*l vestibule or posterior vagin* and anterior vagin*. It
is recommended to avoid the cl*tor*s fossa which contains fragments of keratinizated cells which
can be confused with the superficial epithelial cells obtained from the during the estrus period.
Once the rod inside the vagin* the vet tehnician makes movements of rotation in the opposite
direction to the direction of rotating the cotton wool. One should be aware of this fact because there
is a risk of cotton wool to get detached from the rod. The sample taken was displayed on a glass
blade by rolling over the wool part of the rod. The smearl dyeing was made using the Diff Quick
method. The examination of cytovagin*l smear was done by help of the Leica microscope owned
by CLC Horia Cernescu.
Preparing the animal for the blood sampling began with shaving the middle area of the
forearm and antisepting it with betadine solution 4%. The blood samples for determining the
progesterone level in blood was done by stinging a needle attached to a syringe into the vena
cefalica antebrahiala. A quantity of 1- 2.5 ml of blood was taken.
The blood taken was transferred into a vacuum container which does not contain
anticoagulant substances . The container was labelled with the owner and pet IDs. The sample thus
obtained was sent, as soon as possible, at Bioclinica laboratory. The determination of the
progesterone level was done by means of the chemiluminiscence method, using the Siemens Advia
Centaur device. The value obtained was expressed in mmoles/l and nanograms per milliliter.
Preparing the female for the ovaries ultrasound examination consisted in trimming or
shaving the flank area , covering the transverse processes of the lumbar vertebrae 3 and 4. The
female will be positioned in lateral or dorso-lateral decubitus, either left or right depending on the
ovary to be viewed. There are some situations in which, due to the presence of certain portions of
the intestine in the proximity of the ovary, viewing it is more difficult. In such situations, the
females can be examined in upright position. It is worth mentioning that the animal being shaved
is absolutely mandatory in order to have good quality images, so it should not be left to the
discretion of the owner. Many owners refuse to have the animal shaved, due to aesthetic reasons.
The echography of the ovaries should be performed after a 12-hour water diet to have an empty
intestin.
After applying the gel on the freshly shaved area, an attempt was made to visualize the
kidney caudal pole. This represents a landmark in the case of the examination of the ovaries
because they are close to the kidney. The ovary is positioned caudo-laterally towards the distal
portion of the kidney. An anatomical peculiarity of female genital apparatus, namely being
positioned more cranially to the kidney compared to the left kidney. Thus, the right ovary is
positioned more cranially compared to the left one. The echography of the ovaries should start with
the left ovary, since it is easier to locate. It should be also taken into account the fact that the ovaries
are located superficially, very close under the skin. The ultrasound examination was done using a
My Lab 50 Esaote, Betford Hills, NY, USA apparatus equipped with a 3-9 MHz frequency convex
probe set to mode B, property of CLC Horia Cernescu..
Results and discussions
Please note that the study has been negatively impacted by a number of factors such as:
the reduced availability of owners to be present at the clinic following the shedule established, the
anxiety of the bitches that caused tachypnea and the tissues, and the hollow/cavitary organs
containing gas.
348
The Bucovina Shepherd beatch was investigated twice in a span of two days. At the first
medical investigation the progesterone level was 1.9 ng/ml and the image of the cyto-vagin*l
smeard was impossible to be interpreted due to the abundant presence of the estrus mucus. The
second investigation showed that the superficial cells with no nucleus of all keratenized cells
cheratinizate were around 72%. According to data in the scientific literature , at the percentage of
cells without nucleus of 72 per cent, the female is supposed to be on the everge of ovulation, but
the progesterone level of 2.5 ng/ml does not indicate this fact. The data in the literature show a
very large range (4-10ng/ml) of the progesterone levels at the time of ovulation. Related to the
percentage of cells with no nucleus, ENGLAND (3) consider that it is supposed to be 75, at the
ovulation moment. This demonstrates that the percentage of the keratenized cells in the cyto-
vagin*l smear used to determine the moment of ovulation is not a good method.
Concerning the Labrador Retriever beatch, the oestrus cycle showed a percentage of 16
cells with no nucleus. The ovary follicles had an even appearence, with thickened walls, feature
specific to the follicules before ovulation. Thus, in this situation, the two medical investigation
collate.
The Tibetan Mastiff female was investigated according to the cyto-vagin*l smear, the
progesterone level and through the ultrasound method. In this particular situation, there is no
discrepancy, meaning that the ultrasound results, the percentage of the superficial cells with no
nucleus and the progesterone levels showed that the famale did not ovulate. The sonograph pointed
that on the ovary monitoredthere are 6 follicles.
Tabel 1. The connection among the oestrus cycle, the P4 level and the
ultrasound results in a West Highlander White Terrier bitch
Data The cyto-vagin*l smear/The
procentage of the superficial no
nucleus cells
The ultrasound appearance of
the ovaries aloare nivel
P4
10.11.2015
1
1,1 ng/ml
Unplayabal
2,3%
349
3.11.2015
0,73 ng/ml
7.11.2015
2
2,59 ng/ml
9.11.2015
6,4ng/ml
The West Highlander White Terrier female was investigated, using all the three methods,
4 times, every 2-4 days, according to its evolution concerning the sexual cycle. Even if the first
cyto-vagin*l smear was impossible to interpret (Table 1), speaking from the cytology point, the
female had an ascending evolution regarding the cells without a nucleus. The same trend was found
in the progesterone level. The beatch was at a basic/basal level (values between 0.73 ng/ml and
2.59 ng/ml) during the first three days of the investigation. On the last day, it was noticed that the
progesterone value of 4ng/ml (value which most studies consider is an indicator for ovulation )
collated neither with theultrasound image of the ovaries, nor with the vagin*l cytology, . We would
have expected that at a progesterone value of 6,4 ng/ml, the procentage of the cells with no nucleus
to be 75%, (ENGLAND and LEVY) (2, 3) and the follicles to be well-defined.. Ultrasound follicles
appearance was, however, one specific to the preovulation follicles.(Table 1). This reveals at least
ESTRUS
4
8,54%
ESTRUS
3
5%
52,3%
ESTRUS
350
a fact, namely, at a progesterone value of 6/ml, 4ng , the female did not ovulate. We consider
that, out of the three methods of investigation, the ovaries ultrasound exemination brings the most
precious information about the time of ovulation. The data in the scientific literature on
progestrerone level at which ovulation occurs, are vastly different ranging from 4-10 ng/ml (5).
Table 2. Connection among estrous cycle, the P4 level and the ovaries
ultrasound appearance in a Bichon Maltese bitch
Data The cyto-vagin*l
smear/The procentage of
the superficial no nucleus
cells
The ultrasound appearance of the
ovaries
P4 value
ng/ml
3.06.2016
_
_
-
5.06.2016
__
-
_
_
6.06. 2016
2,52
Proestrus
4%
1%
Proestru
Proestrus
3%
351
8.06.2016
2,32
10.06.2016
5,39 g/ml
3.06.2016
20,24
ng/ml
The Bichon female was investigated, using all the three methods, six times, every 1-3 days,
according to its evolution concerning the sexual cycle
Speaking from the cytology point, the female had an ascending evolution regarding the
cells without a nucleus. From one medical exam to another, the percentage of the cells with no
progressive nucleus grew. According to Table 2, when the cells with no nucleus were 23,68%, the
progesterone level was 5,39 ng/ml, very close to ovulation time.. On the penultimate day of the
investigation the female was exemined only hormonally and cytologically, because we had no
access at the ultrasound apparatus at the weekend. On the last day of the examination, the female
had a smear specific to the metestrus period beginning, and the P4 level was 20,24 ng/ml.
The data in the scientific literature show that considering the percentage of cells with no
nucleus out of all keratenized cells in order to determine the ovulation time, envolves great
difficulty and it is subjected to errors of interpretation. This fact is due to the cyto-vagin*l smear
being uneven. Thus, the percentage of keratinizated cells with no nucleus differs from one image
to another.
Estrus
8,84%
Estrus
Metestru
Metestrus
352
The evolution of the progesterone levels was normal relative to the folllicular
development and cytology. Thus, the female had a basal/ basic P4 level until about 3 days before
ovulation. About 2 days after ovulation the P4 level was 20.24 ng/ml. The P4 value in a certain
period of time after ovulation differs greatly from one bitch to another and it might be due to the
number of ovarian follicles and to the number of corpus luteum. Thus, a larger number of corpus
luteum will cause a higher and sudden progesterone level.
In terms of the ultrasound results, the ovarian follicles had an ecogenous appearance to
the last medical examination . Since the last examination, a change in the ovaries appearance
could be noticed- they turned hypo-ecogenuous. Also, the ovaries form evolved from a round shape
at the beginning of the monitoring (A ', C ') to elliptical form (D ') at the end of it.
We noticed that the females belonging to large breeds and the obese ones, the exemination
using the echography/ultrasound apparatus is performed with greater difficulty, situation reported
also in the scientific literature (5). The easiest ultrasound examination can be done during the
follicular sexual cycle. (proestrus and the estrus). During the proestrus cycle, the ultrasound
appearance of the ovaries show regular and ecogenous shape, having 6-9 mm, with very thin
follicula wall. The follicles grow progressively from the early follicular phase until its end. With
the approaching of ovulation time, the preovulation follicle thickens its wall up to 1mm. According
to the informtion in the literature a preovulating follicle can change its shape becoming flat,
situation that the current study did register. At the ovulation time the follicular cavity dissapear
(follicular collapse).
There is information in the literature that in half of the cases, after ovulation, soem hypo-
ecogenous sstructures are still be found inside the ovary (8). These structures are different from the
preovulation follicles being smaller and irregularly shaped (9).
It is rather difficult to assess the number of follicles according to the literature (5). These
researchers notice that 45 % of the follicles do not ovulate. These round ecogenous structures can
be seen up to three days after ovulation. This situation may mislead an inexperienced examiner.
Another structure that may mislead the vet is the liquid present between the ovary and ovarian
bursa, situation that can occur in 40% of cases (1, 4). A day after the ovulation, there is a blood
accumulation in the follicular cavity that leads to the appearance of the hemorrhagic corpus
luteum. They have a structure similar to the preovulation . It is therefore very important that the
ovaries examination to take place daily in order to show the exact ovulation moment.
According to LEVY and FONTBONNE (5), detecting the ovulation moment by
ultrasound investigation is just 10 percent more accurate compared to the progesterone level
determination. Setting the ovulation moment is imperiously needed especially if the artificial
insemination is done with chilled or frozen sem*n, or if the animal is susceptible of infertility (6).
The bitch ovaries are difficult, and sometimes impossible to be examined through
ultrasonography, because they are small, difficult to be differentiated from the surrounding tissues
and are often obscured by the intestinal gas (3). The rapid disappearance of the follicular cavity,
correspondent to the ovulation , was detected only in two bitches. Although ultrasound changes
during the estrous cycle were well-studied, the exact moment of ovulației cannot be predicted (6).
Conclusions
Examining the ovaries in the follicular phase of the sexual cycle by ultrasound device is
easier in the situation of a small or medium –sized female than a large one.
The ultrasound examination is the most certain method out of the three ones described.
Even if the sudy faced some non-ideal situations, we can conclude that the ovaries
ultrasound examination is a valuable method to predict the ovulation moment in bitches.
353
To optimize the results of determining the ovulation moment it is recommended to collate
the ultrasound examination with at least one of the other two methods of investigation.
References 1. DOW, C. (1990) - Ovarian abnormalities in the -Journal of Comparative Pathology70,59-69. 2. ENGLAND, G.C., CONCANNON, P.W. (2003) -Determination of the optimal breeding time in the :
basic considerations.In: Recent advances in small animal reproduction. Ithaca, NY: International Veterinary Information Service,2003 Disponible en www.ivis.org.
3. ENGLAND, G.C., YEAGER, A., CONCANNON, P.W. (2003) - Ultrasound imaging of the reproductive tract in the . In: recent advances in small animal reproduction. Ithaca, NY: International Veterinary Information Cervice.
4. GINTHER, O.J. (2007) - Ultrasonic imaging and Animal reproduction-color-doppler Ultrasonography
5. LEVY, X., FONTBONNE, A. (2007) - Determining the optimal time of mating in es:particularities-Rev Bras Reprod Anim, Belo Horizonte, v.31, n.1, p.128-134, jan./mar. 2007. Disponível em www.cbra.org.br.
6. MCENTEE, K. (1990) - Cysts in and around the ovary-In Reproductive Pathology of Domestic Mammals-San Diego,Academic Press, pp52-68
7. NYLAND, T.G., MATTOON, J.S. (2002) - Small Animal diagnostic ultrasound, second edition 8. RASKIN R.E., MEYER D.J(2001) - Atlas of canine and feline cytology. Philadelphia, WB Saunders
co, 2001, pp. 277-312. 9. VERMEULEN, M.A.E. (2009) - Ovarian color-Doppler ultrasonography to ovulation in the -
Research project veterinary Medicine Lousiana State University.
354
Effect of rozmarinic acid supplementation on in vitro maturation
of bovine oocytes
Simona MARC, Camelia TULCAN, Oana BOLDURA, A. SOLONAR, G. OTAVĂ, G. GODJA,
I. HUȚU, C. MIRCU University of Agricultural Sciences and Veterinary Medicine of Banat “The King of Michael I” of
Romania, Faculty of Veterinary Medicine, Timisoara, Romania [emailprotected]
Abstract Antioxidants supplementation of in vitro culture media exerts the key role to reduce the effects of
reactive oxidative species produced during assisted reproduction technique. The objective of the study was
to determine the effect of rosmarinic acid addition to the in vitro culture media on bovine oocytes maturation
rate based on morphological changes. Bovine COC’s were matured according to their morphological class
(class I, II and III) in two groups: control (M) and supplemented with rosmarinic acid (105 µM, AR) in TCM
199 HEPES modification media at 38.50C in 5% CO2 humidified air atmosphere for 24h. Comparing the
groups, relative to the number of COC’s matured, a increase in their maturation features is observed, with
26.81% % (AR1), 21.67% (AR2) and 23.34% (AR3), respectively in groups supplemented with rosmarinic
acid. The oocyte class is associated with their capacity to develop in vitro based on their morphological
examination.
Key words: antioxidants, oocyte, rosmarinic acid
Introduction
In vitro fertilization (IVF) is an assisted reproduction technique (ART) used with good
results in bovine reproduction, with 443.533 of bovine embryos obtained worldwide in the year
2012 according to statistics of the International Embrio Transfer Society
(http://www.iets.org/pdf/comm_data/december2013.pdf)
Successful ART is influenced by many factors, among which reactive oxygen species
(ROS) has a significant role (Agarwal et al., 2014).
Sources of ROS during ART procedures could be either endogenously (immature
spermatozoa, leukocytes, oocyte, cumulus mass cells, follicular fluid, embryos) or exogenous
environmental factors (visible light, culture media, pH, temperature, oxygen concentration,
centrifugation, cryopreservation)(Agarwal et al., 2014).
Reproductive systems possess antioxidant defense mechanisms that maintain equilibrium
between pro- and anti-oxidants (Roychoudhury et al., 2017; Agarwal et al., 2014); but during in
vitro conditions, the gametes needs to be protected by supplemented antioxidants.
Studies indicates that supplementing maturation media with different antioxidants such as
β-mercaptoethanol (Sadeesh et al., 2014), cysteamine (Beheshti et al., 2011); cysteine (Mircu et
al., 2015), vitamin C (Sovernigo et al., 2017; Comizzoli et al. 2003; Agarwal et al., 2014), plant
antioxidants – flavonoids (Kang et al., 2016, Mbemya et al., 2017) can improve oocytes maturation
based on nuclear morphological changes and on gene expression.
Another natural antioxidant used in ART, especially in freezing extenders were improves
sperm quality after cryopreservation, is rosmarinic acid (Malo et al., 2010; Luno et al., 2014; Luno
et al., 2015; Olaciregui et al., 2017).
Rosmarinic acid is an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid derived from
hydroxycinnamic acid, that belongs to polyphenols group and is found as an active compound in
several medicinal plants (Rosmarinus officinalis, Salvia officinalis, Mentha arvense, Ocimum
basilicum, Thymus vulgaris etc) (Krajcovicova et al, 2013).
355
Rosmarinic acid has antiinflamatory, antiviral, antibacterial, antimutagen, antidepressant,
antiallergic, antioxidant effects. His antioxidant activity is supporting by enhancement of
superoxide and hydroxyl scavenging (Krajcovicova et al, 2013).
Hajhosseini et al. (2013) observed that rosmarinic acid has a preventive effect on Sertoli
cells apoptosis caused by electromagnetic fields.
Although in literature are data regarding beneficial effects of green tea polyphenols (Wang
et al., 2007; Ly et al., 2015) and catechins (Roychoudhury et al., 2017) on reproductive health and
on IVF parameters and subsequent development, there are no studies, to our knowledge, regarding
the effect of rosmarinic acid supplementation on bovine oocytes in vitro maturation. For this reason
the purpose of this present research was to evaluate the effect of rozmarinic acid added in media
for in vitro bovine oocytes maturation on their maturation rate based on morphological changes.
Materials and methods
Bovine ovaries (n=18) were collected from local slaughterhouse and transported to the
laboratory in containers containing 0.9% NaCl solution supplemented with antibiotics (Pen/Strep),
at 33-350C within two hours. Handling medium for COC (cumulus -oocytes- complexes) was
Dulbecco-PBS (D-8662) supplemented with 100 µl Pen/Strep (17-602F, Lonza); 3.6 mg sodium
piruvate, 30 mg BSA (A9647, Sigma-Aldrich), 100 mg glucose (G7021, Sigma-Aldrich). COCs
were aspirated by puncture procedure from medium to large follicles with 18G needle attached to
a 5 ml syringe.
Classification of COCs based on morphological aspects was done under stereomicroscope
(Stemi 2000-C, ZEISS) with hot plate (33.40C): Ist class - CI (COCs with cumulus compact and
unexpanded, with full or at least 5 layers of cumulus cells, cytoplasm clearly seen, dense and
hom*ogenous, IInd class – CII (COCs with cumulus compact, thick, 2-4 layers of cumulus cells,
covering all of zona pellucida, cytoplasm dense, with uniform granulation) and IIId class - CIII
(oocytes partially denuded of cumulus cells, or with 1-2 complete layers of cumulus cells and/or
with irregular shrunken cytoplasm).
The maturation culture medium was prepared in our laboratory after Parrish et al. (1986)
protocol with minor modifications: TCM 199 HEPES modification media, (M2520) with 10% ECS
and 15 µl FSH (F8174, Sigma-Aldrich) - group M (control), in experimental group we added
rosmarinic acid (105 µM)(536954, Sigma Aldrich) - group AR. Pools of 8-10 COCs were
maturated in 400µl media in 4 well dishes (Nunc, Germany) covered with mineral oil at 38.50C in
5% CO2 humidified air atmosphere for 24h. After 24h of culture, all COC were examined for
maturation, signs as expansion and mucification of cumulus cells were observed. The COC’s were
maturated according to there their morphological class (M1, M2, M3, AR1, AR2, AR3).
Results and discussions
The results of supplementation of in vitro media with rosmarinic acid on bovine oocytes
morphological aspects are presented in figure 1 and 2.
After in vitro maturation of cow's oocytes in the medium without antioxidants (M group)
we noticed at the morphological assessment that 55% of class I COCs (M1), 53.33% of class COC
II (M2) and 26.66% of class III COCs (M3) were matured. In the rosmarinic acid supplemented
groups (AR group), 81.81% of class I COCs (AR1) were matured after 24 hours, 75% of class II
(AR2) and 50% of class III (AR3).
356
Figure 1. Morphological evaluation of bovine COC’s before and after IVM
Comparing the groups, relative to the number of COC’s matured, a increase in their
maturation sign is observed, with 26.81% % (AR1), 21.67% (AR2) and 23.34% (AR3),
respectively. Regardless of the treatment applied, the oocyte class is associated with their capacity
to mature in vitro based on their morphological examination.
Figure 2. Aspects of COC’s classification according to their morphological class and
experimental group before IVM (A – Ist class, B – IInd class, C – IIId class), after IVM, group M
(a, b, c), group AR (d, e, f)(5X)
Morphological evaluation of bovine COC's during IVM
5
10
15
20
25
30
35
Before IVM After IVM Before IVM After IVM Before IVM After IVM
C1 C2 C3
Morphological class
CO
C's
nu
mb
er
M
AR
357
These results are sustained also by BAX/BCL2 gene expression (unpublished data), where
we observed that BCL-2 (anti-apoptotic gene) had higher levels in Ist class COC’s from rosmarinic
acid (AR1) groups compared with the other groups and BAX (pro-apoptotic gene) level is
indirectly proportional with the quality of the oocyte, with the highest level in IIId class oocytes,
what it means that both antioxidant supplementation and the quality of the oocyte has an important
role in maintaining cellular viability.
Oxidative stress has negative effects on in vitro gametes and embryos (Agarwal et al.,
2014; Beheshti et al, 2011) and excessive ROS production can’t be controlled properly by the
mammalian cells antioxidant systems (superoxide dismutase, glutathione system, thioredoxin
system, catalase, thiol compounds) that scavenge ROS or prevents its formation due to the multiple
potential sources of ROS, lack of physiological defense mechanisms etc (Sadeesh et al., 2014; Lu
et al., 2013; Agarwal et al., 2014). That’s why it’s important to add antioxidants in media used in
ART procedures (Sadeesh et al., 2014; Beheshti et al., 2011; Mircu et al., 2015; Sovernigo et al.,
2017; Kang et al., 2016, Mbemya et al., 2017).
From literature data we know that rosmarinic acid antioxidant effects protects ovine
spermatozoa during lyophilization by maintaining the sperm DNA integrity and after reconstitution
of the freeze-dryed spermatozoa, they can sustain fertilization and even embryonic development
(Olaciregui et al., 2017). Also in boar sem*n cryopreservation rosmarinic acid it is used as an
antioxidant where improves the post-thaw quality of spermatozoa and the ability to fertilize (Malo
et al., 2010; Luno et al., 2014; Luno et al., 2015).
Our preliminary results suggests that antioxidant properties of rosmarinic acid is effective
also on bovine oocytes in vitro maturation. Further studies are needed to clarify the effects of
rosmarinic acid used during IVM on further steps of IVF technique.
Conclusions
Supplementation of the cow's oocyte culture media with rosmarinic acid can determine a
higher quantity of bovine oocytes matured in vitro based on morphological evaluation.
Quality of the COC used for in vitro techniques has an important role in the success of the
experiment.
Aknowledgments
The research was carried in the IVF (In Vitro Fertilization) laboratory from the Horia
Cernescu Research Unit equipped through POSCCE 2669 program
References
1. AGARWAL, A., DURAIRAJANAYAGAM, D., PLESSIS, S. (2014) - Utility of antioxidants during assisted reproductive techniques: an evidence based review, Reproductive Biology and Endocrinology, 12, 112, https://doi.org/10.1186/1477-7827-12-112.
2. BEHESHTI, R., MOHAMMADI-ROSHANDEH,A., GHALEHKANDI GIASI, J., GHAEMMAGHAMI, S., HOUSHANGI, A.F. (2011) - Effect of antioxidant supplements on in vitro maturation of bovine oocyte,
Advances in Environmental Biology, 5, 7, 473-1475. 3. COMIZZOLI,P., WILDT,D.E. PUKAZHENTHI, B.S. (2003) - Overcoming poor in vitro nuclear maturation
and developmental competence of domestic cat oocytes during the non-breeding season, Reproduction, 126, 809-816.
4. HAJHOSSEINI, L., KHAKI, A., MERAT, E., AINEHCHI, N. (2013) – Effect of rosmarinic acid on Sertoli cells apoptosis and serum antioxidant levels in rats after exposure to electromagnetic fields, Afr J Tradit Complement Altern Med., 10, 6, 477-480.
5. KANG, J.T., MOON, J.H., CHOI, J.I., PARK, S.J., KIM, S.J., SAADELDIN, I.M., LEE, B.C. (2016) - Effect of Antioxidant Flavonoids (Quercetin and Taxifolin) on In vitro Maturation of Porcine Oocytes, Asian Australas. J.Anim.Sci., 29, 3, 352-358.
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6. KRAJCOVICOVA, ZDENKA, MELUS, V. (2013) – Bioactivity and potential health benefits of rosmarinic acid, University Review, 7, 2, 6-14.
7. LU, J., HOLMGREN, A.(2013) – The thiroredoxin antioxidant system, Free radical biology and medicine, 66,8
8. LUÑO V, GIL L, OLACIREGUI M, GRANDIA, j., ANSO, T., DeBLAS, I (2015) - Fertilisation rate obtained with frozen-thawed boar sem*n supplemented with rosmarinic acid using a single insemination timed according to vulvar skin temperature changes, Acta Veterinaria Hungarica, 63, 1, 100-109.
9. LUÑO V, GIL L, OLACIREGUI M, GONZÁLEZ N, JEREZ RA, DE BLAS I. (2014) - Rosmarinic acid improves function and in vitro fertilising ability of boar sperm after cryopreservation, Cryobiology, 69, 1, 157-62.
10. LY, CHRISTINA, YOCKELL-LELIEVRE, JULIEN, FERARO, Z.M., ARNASON, J.T., FERRIER, J., GRUSLIN, A. (2015) – The effect of dietary polyphenols on reproductive health and early development, Human Reproduction Update, 21, 2, 228-248.
11. MALO, C., GIL, L., GONZALEZ, N., MARTINEZ, F., CANO, R., DE BLAS, I., ESPINOSA, E. (2010) – Anti-oxidant supplementation improves boar sem*n characteristics and fertility after cryopreservation: comparison between cysteine and rosemary (Rosmarinus ooficinalis), Cryobiology, 61, 1, 142-147.
12. MBEMYA, G.T., VIEIRA, L.A., CANAFISTULA, F.G., LOIOLA PESSOA, O.D., RIBEIRO RODRIGUES,A.P. (2017) – Reports on in vivo and in vitro contribution of medical plants to improve the female reproductive function, Reprod Clim, (http://dx.doi.org/10.1016/j.recli.2016.11.002)
13. MIRCU C., BOLDURA OANA, IGNATIADI AIKATERINI, RAțIU ANA-MARIA, HUțU I., POPESCU SORINA, TULCAN CAMELIA, AHMADI MIRELA, BONCA GH., MILOVANOV CORNELIA (2015) – Effect of cysteine supplementation on sow cumulus cells and on BCL2 gene expression during in vitro maturation, Lucr Stiin Med Vet USAMV Iasi, Ed.”Ion Ionescu de la Brad”, 58, 292-297.
14. OLACIREGUI, M., LUNO, VICTORIA, DOMINGO, PAULA., GONZALEZ, NOELIA, GIL, LYDIA (2017) – In vitro developmental ability of ovine oocytes following intracytoplasmatic injection with freeze-dried spermatozoa, Scientific reports, 7, 1096, DOI:10.1038/s41598-017-00583-0,
www.nature.com/scientificreports. 15. PARRISH J.J., SUSKO-PARRISH J.L., LEIBFRIED-RUTLEDGE M.L., CRITSER E.S., EYESTONE
W.H., FIRST N.L. (1986) -Bovine in vitro fertilization with frozen-thawed sem*n, Theriogenology, 25, 591–
600. 16. ROYCHOUDHURY, S., AGARWAL, A., VIRK, G., CHO, C-L., (2017) - Potential role of green tea
catechins in the management of oxidative stress-associated infertility, http://dx.doi.org/10.1016/j.rbmo. 17. SAADESH, E., SHAH, F., BALHARA, A.K., THIRUMARAN, S. M. K., YADAV, S., Yadav, P. S., (2014) -
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359
Evaluation of sows oocytes viability through Trypan Blue
staining after vitrification
Simona MARC, C. MIRCU, Nicoleta CREȚAN, G. OTAVĂ, Camelia TULCAN, I. HUȚU
University of Agricultural Sciences and Veterinary Medicine of Banat “The King of Michael I” of Romania, Faculty of Veterinary Medicine, Timisoara, Romania
E-mail address: [emailprotected]
Abstract
Along sperm and embryo cryopreservation that are used routinely also in animal assisted
reproduction, studying are done also on animal oocyte cryopreservation in order to find the best conditions
to preserve their viability. Vitrification is one of the methods that can be used in order to preserve oocytes.
The higher reactive oxygen species that are formed during in vitro conditions can influence the success of
assisted reproduction technique. The aim of this study was to evaluate the antioxidant potential of ascorbic
acid (0.5mM) and rosmarinic acid (105µM) added in media for in vitro maturation on swine oocyte subjected
to vitrification. COC’s viability after vitrification was evaluated by 0.02% Trypan Blue staining. Comparing
experimental groups C (vitamin C) and AR (rosmarinic acid) with group M (control), relative to the number
of vitrified oocytes, a slight increase in their viability is observed, with 16.67% (C, class I) and 33.33% (AR,
class II), respectively. Regardless of the treatment applied, the oocyte class is associated with viability (p =
0.048). Due to low number of oocytes used in each group we can concluded that supplementation of oocyte
maturation media before vitrification with rosmarinic acid and ascorbic acid could produce a slight increase
in viability.
Key words: antioxidants, oocyte, vitrification
Introduction
Vitrification of oocytes and embryos is a revolutionary cryopreservation technique used
both in human (Konc et al. 2014) and in animals (Somfai et al. 2014; Spricigo et al., 2015; Yang
et al. 2002; El-Sokary et al., 2013) assisted reproduction techniques (ART) performed in diferent
conditions and with different results.
Vitrification is defined as the ultrarapid solidification of a solution by an extreme elevation
in viscosity at low temperatures without ice crystal formation (Konc et al., 2014).
The main causes of cell death during cryopreservation is ice crystal formation and toxic
concentrations of solutes. In order to reduce the negative effects of cryopreservation,
cryoprotective additives (CPA) are used. They are: intracellular/membrane-permeating
(propyleneglycol, dimethyl sulfoxide, glycerol, ethylene glycol) and extracellular (sucrose,
trehalose, glucose, amid, ficoll, proteins and lipoproteins). The first ones displaces water via an
osmotic gradient and partly occupies the place of the intracellular water, while the extracellular
cryoprotective additives increases the extracellular osmolarity generating an osmotic gradient
across the cell membrane supporting the dehydration of the cell before cryopreservation and also
prevents the rapid entry of water into the cell after thawing. During vitrification cells are dehydrated
before the ultrarapid cooling by high concentration of CPA (Konc et al., 2014; Yang et al., 2002).
Oocytes are very sensitive to cryoprotectants used during cryopreservation protocols.
Although vitrification of matured porcine oocytes has high survival rates, obtaining embryos by
IVF or ICSI from them is difficult (Somfai et al., 2014). Cooling/warming processes from
vitrification technique of porcine oocytes at MII stage determined accumulation of reactive oxygen
species (ROS), parthenogenetic activation and spindle abnormalities (Somfai et al., 2014).
Sources of ROS during ART procedures could be either endogenously (immature
spermatozoa, leukocytes, oocyte, cumulus mass cells, follicular fluid, embryos) or exogenous
360
environmental factors (visible light, culture media, pH, temperature, oxygen concentration,
centrifugation, cryopreservation)(Mbemya et al., 2017; Agarwal et al., 2014). When is an
imbalance between reactive oxygen species (ROS) and a biological system’s ability to readly
detoxify the reactive intermediates or repair the resulting damage, oxidative stress apears
(Roychoudhury et al., 2017).
Because during in vitro conditions oocytes are separated from the body and do not benefit
from maternal antioxidant protection, supplemented antioxidants are needed. Studies indicates that
supplementing maturation media with different antioxidants such as β-mercaptoethanol (Sadeesh
et al., 2014), cysteine, cysteamine (Beheshti et al., 2011), palm pollen grain extract (Salek-
Abdollahi et al., 2015), quercetin and taxifolin – exogenous flavonoids (Kang et al., 2016),
Gundelia Tourneforii leaves hydro alcoholic extract (Abedi et al., 2014) can improve oocytes
maturation based on nuclear morphological changes.
Regarding vitamin C (L-ascorbic acid), a water-soluble antioxidant there are few studies
to investigate it’s antioxidant effects during in vitro maturation of oocytes (Sovernigo et al., 2017;
Comizzoli et al. 2003; Tatemoto et al., 2001), most of studies were performed to emphasize it’s
beneficial role on freezing spermatozoa (Varo et al., 2014; Fanaei et al., 2014) or on improved
motility and reduced DNA damage in post-thaw spermatozoa.
Another antioxidant used especially in freezing extenders which improved sperm quality
after cryopreservation was rosmarinic acid (Luno et al., 2014; Luno et al., 2015; Olaciregui et al.,
2017). Rosmarinic acid is one of the first secondary metabolites produced in plant cell cultures in
extremely high yields, up to 19% of the cell dry weight. Other promising biological activities of
rosmarinic acid and its derivatives (rabdosiin and lithospermic acid B) are: improvement of
cognitive performance, prevention of the development of Alzheimer's disease, cardioprotective
effects, reduction of the severity of kidney diseases and cancer chemoprevention. (Bulgakov et
al.,2012).
The purpose of this present research was to evaluate the viability of swine oocyte after in
vitro maturated in media supplemented with ascorbic acid and rosmarinic acid and cryopreserved
through vitrification.
Materials and methods
Swine ovaries (n=10) were collected from slaughterhouse and transported to the laboratory
in containers containing 0.9% NaCl solution supplemented with antibiotics (Pen/Strep), at 33-350C
within two hours. Handling medium for COC (cumulus -oocytes- complexes) was Dulbecco-PBS
(D-8662) supplemented with 100 µl Pen/Strep (17-602F, Lonza); 3.6 mg sodium piruvate, 30 mg
BSA (A9647, Sigma-Aldrich), 100 mg glucose (G7021, Sigma-Aldrich). COCs were aspirated by
puncture procedure from medium to large follicles with 18G needle attached to a 5 ml syringe.
Classification of COCs based on morphological aspects was done under stereomicroscope
(Stemi 2000-C, ZEISS) with hot plate (33.40C) after criteria of Antosik et al. (2010) with minor
modification: Ist class - CI (COCs with cumulus compact and unexpanded, with full or at least 5
layers of cumulus cells, cytoplasm clearly seen, dense and hom*ogenous, IInd class – CII (COCs
with cumulus compact, thick, 2-4 layers of cumulus cells, covering all of zona pellucida, cytoplasm
dense, with uniform granulation) and IIId class - CIII (oocytes partially denuded of cumulus cells,
or with 1-2 complete layers of cumulus cells and/or with irregular shrunken cytoplasm).
The maturation culture medium was prepared in our laboratory after Parrish et al. (1986)
protocol with slight modifications: TCM 199 HEPES modification media, (M2520) with 10% ECS
and 15 µl FSH (F8174, Sigma-Aldrich) - group M (control), in experimental groups we added
ascorbic acid (0.5 mM) – group C and rosmarinic acid (105 µM) - group AR. Pools of 8-10 COCs
361
were maturated in 400µl media in 4 well dishes (Nunc, Germany) covered with mineral oil at
38.50C in 5% CO2 humidified air atmosphere for 44h. After 44h of culture, all COC were examined
for maturation, signs as expansion and mucification of cumulus cells were observed and were
cryopreserved by vitrification according to there experimental group (M, C, AR group) and their
morphological class (M1, M2, M3, C1, C2, C3, AR1, AR2, AR3).
Vitrification steps were: 15 min in Freezing 1 media (750 μl DMSO, 750 μl EG, 850 μl
ECS and 7.65 ml TCM99), 1 min in Freezing 2 media (1500 μl DMSO, 1500 μl EG, 1.71g sucrose,
520 μl ECS, 5.23 ml TCM199), aspiration in 0.5 ml straws, sealed with MRSIDUAL V3 device
(IMV) and imersed into liquid N2 container, where they staid for 6 days.
Thawing steps were: 1 min, at 370C in a water bath, 1 min into Thawing 1 media (3.42g
sucrose, 5.28 ml TCM199 and 1.30 ml FCS), 3 min into Thawing 2 media (1.9g sucrose, 6.64ml
TCM199 and 1.65 ml FCS).
COC’s viability after vitrification was evaluated by 0.02% Trypan Blue (T646, Sigma)
staining, for 2 minutes.
Results and discussions
The results of viability evaluation of 57 swine in vitro maturated COCs and after
vitrification, done by Trypan blue staining methods are presented in figure 1 and 2.
Figure 1. Classification of swine COC’s after vitrification based on there viability
After thawing sow oocytes from the control group, 33.33% of the class I (M1) oocytes
were viable and 66.66% non-viable, of the second class (M2) none was viable and in the third class
oocytes (M3), 23.08% were viable and 71.19% were not viable.
Supplementation of the maturation media with vitamin C did not lead to an increase in the
oocytes viability, regardless the morphological classification, thus class I (C1) and II (C2) 50%
were viable and in the third class 33.33% (C3).
Choosing the vitamin C supplement as antioxidant for in vitro maturation media of swine
COCs was based on other researcher’s results. Comizzoli et al. (2003) observed that the
compromised cat oocyte function during non-breeding season can be overridden by including
supplemental FSH and antioxidants (0.5 mM vitamin C or cysteine) in maturation media. Kere et
Viability of swine COCs after vitrification
2
4
6
8
10
12
14
16
18
survived degenerated survived degenerated survived degenerated
C1 C2 C3
Morphological class
No
of
CO
Cs
vitr
ifie
d
M
C
AR
362
al. (2013) testing various concentrations of vitamin C supplemented in IVM and IVC media of
porcine oocytes and parthenotes and handmade cloned embryos observed that although nuclear
maturation of oocytes was not affected by the addition of vitamin C, the intracellular glutathione
levels were significantly increased and ROS reduced at 50 µg/ml vitamin C; added in IVC media,
vitamin C improved blastocyst rates and total cell numbers and reduces apoptotic indices. In
another study done on bovine oocytes, Sovernigo et al. (2017) suggest that antioxidants (vitamin
C 50 µg/ml, quercetin 2µM, cyteamine 100µM, carnitine 0.5mg/ml or resveratrol 2µM) used
during IVM may reduce oxidative stress which improved blastocyst development.
In COC’s groups were we used rosmarinic acid as antioxidant we observed 66.66% of
class I (AR1) and II (AR2) and 55.55% of COCs class III (AR3) survived after vitrification.
Rosmarinic acid is used as an antioxidant in cryopreservation of sem*n. Olaciregui et al. (2017)
proved for the first time that ovine spermatozoa freeze-drying in medium supplemented with 105
µM rosmarinic acid can be lyophilized effectively, stored at room temperature for long term, and
even to starts embryo development after ICSI. Acid rosmarinic (105 µM) has beneficial effects
also on boar sem*n cryopreservation were improves sperm parameters (total and progressive
motility, viability, acrosome integrity) and sperm DNA integrity by reducing DNA oxidation rate
(Luno et al., 2014; Luno et al., 2015). In swine reproduction, Somfai et al. (2014) reported the first
successul piglet production from cryopreserved oocytes by vitrification.
Comparing groups C and AR with group M, relative to the number of vitrified oocytes, a
slight increase in their viability is observed, with 16.67% (C1) and 33.33% (AR1), respectively.
The results obtained for the experimental groups (M, C and AR) require acceptance of the
null hypothesis (Ho); the results does not support the hypothesis that treatment with antioxidants
supports viability (Krusckal-Willis test, p = 0.605) or, on the contrary, causes mortality -
unviability (Krusckal-Willis test, p = 0.429) of oocytes.
Regardless of the treatment applied, the oocyte class is associated with viability (p = 0.048)
at statistically accepted thresholds. The classification of non-viable oocytes is close to the
significance threshold (Krusckal-Willis test, p =0.059).
Vitrification of oocytes can be done before or after in vitro maturation. Comparing these
moments, Milovanov et al. (2016) concluded that cultivation of oocytes before vitrification brings
more advantages for the meiotic resumption.
Figure.2 Live oocyte from group AR1 (1), dead oocytes from group M3 (2) and oocytes from
group C3, A-dead oocyte, B-live oocyte (3)
Staining methods used in ART are very useful in order to see the quality of gametes and
of embryos obtained. Some of them do not affect the viability of the cell, if are used correctly and
for a short time (Brilliant Cresyl Blue, Hoechst 33342). Tripan blue (adiazo dye) is a supravital
stain and an inexpensive marker that is used for studying cellular viability (membrane of live cell
363
is able to exclude the dye, whereas a nonviable cell will have a blue cytoplasm) (Melzer et al.,
2016).
Testing the viability of the oocytes after cryopreservation is a mandatory step in order to
see if the technique used had good results due to numerous factors implicated.
Further experiments are needed to clarify the effects of antioxidants on viability of the
oocyte during vitrification and their capacity to sustain fertilization and developmental competence
after thawing.
Conclusions
Viability of oocytes depends on their class, while lack of viability is not necessarily
associated with quality classes, so oocytes in C1 group had a higher viability of 16.67%
and those in group AR by 33, 33% of the control group (M1).
Supplementation of oocyte maturation media before vitrification with rosmarinic acid and
ascorbic acid could determine a slight increase in viability, but this can not be supported
statistically by our study.
The use of Trypan blue to study oocytes viability is a quick, easy and efficient method
Aknowledgments
The research was carried in the IVF (In Vitro Fertilization) laboratory from the Horia
Cernescu Research Unit equipped through POSCCE 2669 program
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365
Content of amino acids in blood serum in sows with idiopathic
hipogalaxy
Viorica GURDIS
State Agrarian University of Moldova [emailprotected]
Abstract
Hipogalaxy is the result of some pathological processes occurred in gestation which are manifested
by hormonal deviation and deviation of some metabolic processes. A particular content of amino acids is
necessary for a normal vital activity of the body and an adequate metabolism. By this experiment it was
observed the influence of amino acids over the lactation. The results of amino acids screening in sows attest
that their values in hypogalactic sows are different from those of the animals with a normal lactation by the
reduced content of cysteine and tryptophan in the serum and the content of tyrosine significantly increased.
The achieved results can be indicated for the precocious diagnosis of hypogalaxy.
Key words: hipogalaxy, tryptophan, cysteine, tyrosine, postpartum, lactation.
Introduction
From the physiological point of view the hipogalaxy represents the final result of some
pathological processes during the gestation which are manifested by the hormonal deviation of
hypothalamic-pituitary axis and of some metabolic processes. Keeping a particular balance of
amino acids in the body represents the necessary condition which ensures both the metabolic
balance and the optimal galactopoiesis. That is why keeping the functional stability of metabolic
processes in the body represents the basis without which any complex therapy cannot bring positive
results [5,7,8]
For a normal vital activity of the body and an adequate metabolism it is necessary a particular
qualitative and quantitative content of amino acids. Amino acids in the body constitute the main
layer which ensures the synthesis of proteins, enzymes, purines and pyrimidines, biologically
active compounds of peptide origin and other compounds. If necessary, the amino acids can serve
as the energy source by the oxidation of their carbonic components [9,10].
A special interest in the regulation of vital processes represents the tyrosine which is a semi-
essential amino acid which forms by the hydroxylation of phenylalanine. From the tyrosine there
are synthesized thyroid hormones, it can constitute the glucose source or fatty acids and melanin.
Cysteine, whose predecessor is methionine, participates to the formation of
adrenocorticotropic hormone, insulin and glutathione. Recently it was found that from the cysteine
there is formed cysteamine which has a protective role against the ionized radiation. At its turn,
the cysteine is the precursor of oxytocin hormone which stimulates the contraction of myoepithelial
cells of mammary glands increasing the pressure in the galactofore channels and in this way
facilitates the elimination of milk.
Tryptophan represents an amino acid which serves as the predecessor of serotonin, nicotinic
acid and melatonin. The tryptophan is in the composition of α – lactoalbumin which is in the cells
of mammary glands and participate to the synthesis of lactose as a component (B Protein) of the
enzymatic system which is specific for the lactogenic mammary glands. The insufficiency of
tryptophan is manifested by anemia, female and male sterility and in young people affects the
nervous system in the situation of hereditary disorders their metabolism [9,11].
Metabolic spectrum of amino acids is in direct connection with the functional condition of
cells and it can fully characterize the balance. Our attention was focused on several amino acids
which also influence the lactation.
366
Materials and methods
The researches were made in a farm of pigs where the incidence of hypogalaxy was of 4 %.
In the experiment there were include 10 parturient sows (5 sows with normal lactation and 5
hypogalactic sows) in the 2nd and the 3rd lactation. The animals were classified in two equal groups:
with normal lactation (I group) and hypogalactic sows (II group). The sows were kept in closed
premises and they were fed with feed of complete value according to the technology of 3 phases.
It was performed the clinical examination and there were taken blood samples for biochemical
analyses for broadcasting, diagnosing and treatment of hypogalaxy which can have success only if
the indicators of metabolism are investigated in sows with normal lactation and in hypogalactic
sows.
The animals were fed three times a day, water was distributed through the automate trough,
the food was in correspondence with the condition and physiological need but there were situations
of technological non-compliance (lack of food, feeding with non-correspondent food etc.).
The zoo-hygienic conditions for keeping corresponded to the species and physiological
condition of animals.
Results and discutions
Clinical examination provides complex data, aims for the examination of health condition
of animals. According to the opinion of some researchers for finding the hypogalaxy syndrome in
sows and monitoring the efficacy of the administrated treatment there can also be used successfully
the routine clinical indicators which in direct way reflect multiple processes.
General conditions of sows with normal lactation and hypogalactic sows was satisfactory
and it was confirmed by the normal values of clinical indicators (T, B, P) which varied within the
reference limits, but at the same time in hypogalactic sows it was observed the reduction of appetite,
reduced maternal instinct and no interest towards progeny.
Table 1. Clinical indicators
Groups
n
T(ºC) P (contr./minute)
B (movements/min)
M ± m M ± m M ± m
I
Sows with normal
lactation
10
38,2 ± 0,17
96,3 ± 1,32
26 ± 0,4
II
Hypogalactic sows
65
38,5 ± 0,2
94,24 ± 0,92
25 ± 0,8
The values of body temperature, frequency of heart contractions and breath rate are
represented in Chart no. 1. Animals which were under supervision, as it was mentioned above were
in satisfactory general condition. Body temperature of sows with normal lactation and of
hypogalactic sows in average was bigger with 0,3º C in comparison with those with normal
lactation. We mention that the values of body temperature registered by us for the investigated
sows correspond fully with the information from the literature (1,2).
It is well known the fact that the examination of heart contraction frequency and breath rate
is very important in establishing health condition and body reactivity in different extreme
conditions. In chart no. 1 it is also seen that the value of heart contraction frequency in both groups
367
was within the physiological limits. It was a small difference in the limits of calculation error. The
heart frequency in researched animals of both groups corresponds to the limits established by other
(2, 4).
Respiratory movements in sows of the Ist group were of mixt type, costo – abdominal,
rhythmic, symmetric. The frequency of respiratory movements of these animals on the average,
per group corresponded to the physiological limits and constituted 26 breaths per minute. The
frequency of breath in hypogalactic sows was 25 breaths per minute and it corresponded to the
reference limits. Figures shown in chart no. 1 and the indicators of clinical examination show that
the hypogalaxy does not influence negatively the general condition of animals, values of body
temperature, frequency of heart contractions and breath rate.
The mammary gland in hypogalactic sows had a different aspect. In some sows the
mammary glands were rigid, congested, but with flaccid teat. In others, on the contrary they had
normal sizes and consistence and were sensible, warm when palpated, skin of purple color. When
suckled, pigs suckled actively producing sounds for a long period of time. Often they were trying
repeatedly to suckle in more frequent intervals and after suckling they had not become silent. As
the consequence of the effort to suckle the teats were traumatized. When milking only some milk
drops with normal aspect could be obtained or nothing could be obtained. As the energetic
reserves of pigs were decreasing their attempts to suckle also were decreasing and often they
migrated to the warmer portions of the box. In comparison with hypogalactic sows, the mammary
gland of sows with normal lactation was well developed, turgid condition, slightly sensitive to
palpation and when milked there were obtained 1-2 ml of milk.
The average quantity of milk eliminated after a suckling in sows with normal lactation
constituted on average 216,5 ml. Gravimetry made to hypogalactic sows show that these animals
were secreting and eliminating milk after a suckling on average of 80,67 ml or 2,4 times less in
comparison with sows with normal lactation. The quantity of milk which was eliminated by the
sows with normal lactation during a suckling varied from 182 g to 240 g, and in hypogalactic sows
from 54 g to119 g.
A special role have the biochemical investigations in appreciating health condition, in this
context we established the goal to investigate the level of some amino acids in blood serum of the
sows from those two groups. Data in chart no. 2 shows that in hypogalaxy the level of cysteine
decreases significantly, being 2 times lower in comparison with the sows with a normal lactation
(21,0 ±2,3 µmol/l in comparison with 42,7 ±3,4 µmol/l, P<0,001). In all appearances, in
hypogalaxy normal processes of transforming methionine are disturbed and as a consequence the
synthesis of cysteine is reduced and this fact reduces the synthesis of prolactin and respectively the
lactogenesis decreases.
Table 2. Content of cysteine, tyrosine and tryptophan in sows’ blood
Indicators
Sows with normal
lactation Hypogalactic sows
P
M ± m/ lim M ± m/ lim
Cysteine
(µmol/l) 42,7 ± 3,4
(37 -48) 21,0 ± 2,3
(19 – 25) < 0,001
Tyrosine
(µmol/l) 52,8 ± 3,2
(46 – 63) 80,3 ± 4,1
(73 – 86) < 0,001
Tryptophan
(µmol/l) 39,8 ± 2,6
(36 – 46) 24,5 ± 2,7
(10 – 50) < 0,001
368
The content of tyrosine in blood (chart no. 2) in hypogalactic sows (group II) exceeds with
52 % the value registered in sows with normal lactation (group I). It results that in hypogalaxy the
synthesis speed of thyroid hormones and dopamine who predecessor is tyrosine is significantly
reduced.
A significant place in the nitrogen metabolism is occupied by the content of tryptophan. The
data achieved after our investigations (chart no. 1) show that the level of tryptophan in blood of
hypogalactic sows is with 15,3 µmol/l lower in comparison with those from the I group with a
normal lactation. Thus, we find in hypogalactic sows the absence of correlation between the
dynamics of the tyrosine and tryptophan level in blood, fact which eventually can lead to the
reduction of prolactin secretion in anterior pituitary gland.
Conclusions
1. General condition of hypogalactic sows was apparently satisfactory but at the same time it
was found the reduction of appetite, reduced maternal instinct and no interest towards the
progeny.
2. The result of the screening of amino acids in sows show that their values in hypogalactic
sows are different from those with a normal lactation by: the content of cysteine and
tryptophan in serum is significantly reduced (P ˂ 0,05) and the content of tyrosine is
significantly increased (P ˂ 0,05);
3. Screening of amino acids can be indicated for the precocious diagnosis of hypogalaxy.
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dysgalactia syindrome in sows via a genome-widw association study. Institute of Animal Breeding and Husbandry, Christian-Albrechts-University, Kiel, Germany., 2014.
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10. Mavromichalis I, Parr TM, Gabert VM, Baker DH True ileal digestibility of amino acids in sow's milk for 17- day-old pigs. J Anim Sci. 2001 Mar;79(3):707-13.
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369
Cell growth characteristics of equine synovial fluid stem cells
Emoke PALL1,2, Klementina Katalin PALL2, Cristian CRECAN1, Simona CIUPE1, Mihai CENARIU1, Ioan GROZA 1
1University of Agricultural Sciences and Veterinary Medicine, Faculty of Veterinary Medicine, Cluj-Napoca, Mănăştur, no. 3-5, 400372, Cluj-Napoca, Romania
[emailprotected] 2University of Szent Istvan, Godollo, Hungary
Abstract
Equine mesenchymal stem cells (MSCs) have been isolated from various sources such as:
peripheral blood, bone marrow, adipose tissue, umbilical cord, placenta, Wharton Jelly. Besides these
synovial fluid and synovial membrane represents a promising source of mesenchymal stem cells, which can
be harvested by minimally invasive methods. MSCs from these sources have the potential to self-renew and
differentiate into multiple lineages such as chondrogenic, adipogenic and osteogenic. The aim of our study
was to evaluate the growth characteristics of equine synovial liquid stem cells harvested from the
tarsometatarsal joint during arthroscopic surgery. Samples were collected in a sterile syringe and were
diluted and centrifuged at 1500rpm for 7 min. The supernatant were removed and the cells were resuspended
in propagation medium: Dulbecco’s Modified Eagle’s Medium/F12 (DMEM/F12, Gibco) supplemented with
10% fetal bovine serum (FBS, Gibco) and 1% antibiotic-antimycotic (Sigma-Aldrich). The medium was
changed after 3 days. The proliferation ability, cell doubling number, cell doubling time, daily duplication
rate and clonogenic efficacy was evaluated. Isolated cells exhibited plastic adherence capacity, monolayer
growth, and fibroblast-like morphology, high growth and clonogenic capacity. Our study demonstrated the
characteristics of equine synovial fluid derived stem cells, an ideal candidate for veterinary regenerative
medicine.
Keywords: stem cells, synovial fluid, horse, proliferation, cells growth
Introduction
Mesenchymal stem cells (MSCs) are multipotent precursor cells with self-renewal and
differentiation capacity (Dominici et al. 2006, Pall et al., 2015). MSCs are a promising therapeutic
tool in veterinary medicine (Bahamondes et al., 2017) have been isolated
from different animal species including rodents (Penny et al., 2012), dogs (Guercio et al., 2013,
Bahamondes et al., 2017), horses (Barberini et al., 2014, Pall et al., 2016) and rabbits (Tan et al.,
2013). According to the International Society for Cellular Therapy published in 2006, human
MSCs are characterized by plastic adherence capacity, trilineage differentiation capacity and
expression of surface markers such as CD105, CD90, CD44, CD73, CD79α and are negative for
the expression of hematopoietic markers CD34, CD45 and CD19, CD14, human leukocyte antigen
HLA-DR (Dominici et al., 2006). MSCs isolated from animals are not fully characterized. Equine
mesenchymal stem cells (MSCs) have been isolated from various sources such as: peripheral blood,
bone marrow, adipose tissue, umbilical cord, placenta, Wharton Jelly (Barberini et al., 2014,
Tessier et al., 2015, Pall et al., 2016). Besides these synovial fluid and synovial membrane
represents a promising source of mesenchymal stem cells, which can be harvested by minimally
invasive methods. The aim of our study was to evaluate the growth characteristics of equine
synovial liquid stem cells harvested from the tarsometatarsal joint during arthroscopic surgery.
Material and methods
Samples (n=5) were collected in a sterile syringe during atroscopic surgery. The samples
were harvested after owner’s agreement. The samples were diluted and centrifuged at 1500rpm for
370
7 min. The supernatant were removed and the cells were resuspended in propagation medium:
Dulbecco’s Modified Eagle’s Medium/F12 (DMEM/F12, Gibco) supplemented with 10% fetal
bovine serum (FBS, Gibco) and 1% antibiotic-antimycotic (Sigma-Aldrich). Cultures were
incubated at 37 °C in humidified atmosphere with 5 % CO2. After 72 h, non-adherent cells were
removed and the medium was replaced.
Proliferation ability of equine synovial fluid derived stem cells was determined using
MTT colorimetric assay. A concentration of 1x104cells/well was cultured in a 96-well plate. After
24 h 20 μl MTT (2 mg/ml, Sigma-Aldrich) was added to each well and cultures were incubated at
37 °C for 3 h. The fromazan were dissolved with 100 μl DMSO (dimethylsulfoxide, Sigma-
Aldrich) and the absorbance was measured at 450 nm using a spectrophotometer (Bio-Rad,
Hercules, CA, USA). In order to evaluate the proliferation rate the population doubling time were
assessed. A total number of 1x105 cells/ well were seeded in 24-well cell culture plates. After 24 h
(t24h) the non-adherent and the adherent cells (N0) were counted. 24 h later (t48h) the adherent
cells from three wells were counted (N48h). The doubling time (tD) was calculated according to
the formula: tD = (log 2 × t)/ (log N48h - log N0).
The clonal capacity of cells was evaluated using CFU-F assay. 5x102cells/cm2 was cultured
in expansion medium. After 10 days the cells were fixed with 4% paraformaldehyde for 10 min
and stained with 0.5% crystal violet (Sigma-Aldrich, St. Louis, MO) in 10% methanol for 20
minutes and were examined under phase contrast inverted light microscope (Nikon TS100, Nikon
Instruments, Europe) and the colonies (> 50 cells) were counted. CFU-F efficiency was estimated
according to the formula: CFU-F efficiency = (counted CFU-F/cells originally seeded) × 100.
Results and discussion
MSCs were isolated from harvested from equine tarsometatarsal joint during arthroscopic
surgery. The isolated cells were characterized to confirm their caracteristics such as plastic
adherence, expression of specific surface antigens and differentiation potential. Equine synovial
fluid derived mesenchymal stem cells showed fibroblast-like morphology (fig 1.), adherence to
plastic surface and express the specific surface markers (CD90, CD105, CD44, results not shown
here). The proliferation capacities of cells were evaluated using MTT assay.
Figura 1. Equine synovial fluid derived cell morphology
The isolated cells lines exhibited similar proliferation potential in evaluated period (d1-
D6). Optical density (OD) values at each time were evaluated (fig 2.).
371
Figure 2. Proliferation potencial of equine synovial fluid derived mesenchymal stem cell
PD
S1
S2
S3
S4
S5
S6
2 0
4 0
6 0
Figure 3. Population doubling time in equine synovial fluid derived mesenchymal stem cell lines
Individual assessment at each cell line can be concluded that for line S1 the population
doubling time was 44±1.01h, 42±5.8 in S2, 38.33±13.86 h in S3, 41.00±3.00 in S4, 41.66±2.08 in
S5 and 41.66±4.16 in S6 (fig. 3). The average of population-doubling time (PD) was 41.44 ± 1.83 h.
The clonogenic potential of MSCs was assessed by CFU-F assay. Isolated cells lines
displayed colony-forming ability; the frequency of colony forming cells for S1 was 37.66±6.42
colonies/100 cm2, 36.66±6.42 colonies/100 cm2 in S2, 36.33±7.57 colonies/100cm2 in S3,
38.66±4.04 colonies/100cm2 in S4, 35±4.35 in S5 and 37±4.58 colonies /100cm2 in S6 (fig 4.).
CF
U-F
S1
S2
S3
S4
S5
S6
1 0
2 0
3 0
4 0
5 0
Figure 4.The average of colonigenic potential (CFU-F assay) of equine synovial fluid derived
mesenchymal stem cell
OD
45
0n
mCell lines
D1
D2
D3
D4
D5
D6
372
Multipotent mesenchymal stem cells (MSCs) represent a promising source of cells for
regenerative medicine therapeutic approaches in both human and veterinary medicine given their
properties (Singer et al., 2011, Carrade et al., 2013, Clark et al., 2016, Pall et al., 2016). MSCs can
be defined by their morphology, expression of a panel of cell surface markers and their tri-lineage
differentiation (Dominici et al., 2006, Clark et al., 2016). In our study, cells from synovial fluid
were evaluated as possible sources of MSCs, for equine regenerative medicine. The horse is a
valuable species for evaluating the usefulness and efficiency of MSC treatment (Pezzanite et al.,
2015). MSCs from different sources are used, but most often autologous bone marrow-derived
MSCs are used to treat musculoskeletal disorders including tendonitis, osteoarthritis, cartilage
damage, and meniscal injuries (Schnabel et al., 2009, Frisbie and Smith 2010, Caniglia et al., 2012,
De Schauwer et al., 2013, Pezzanite et al., 2015).
MSCs also can modulate endogenous tissue and immune cells (Parekkadan et al., 2010)
and can migrate to the site of injury after receiving specific signals (Chen et al., 2011). MSCs
also can be subject to cryopreservation with minimal loss of potency (Lee et al., 2005),
for future heterologous or autologous transplantation. Numerous studies and clinical trials have
demonstrated the role of MSCs, but further studies are needed to elucidate the exact therapeutic
mechanisms.
Conclusions
Our study showed that mesenchymal stem cells could be successfully isolated form
synovial fluid. Characterization of these cells can be achieved based by their morphology,
immunophenotype and differentiation potential. Results from our study demonstrate the
proliferative and clonogenic potential of equine synovial fluid derived stem cells, and can serve as
a potential source of mesenchymal stem cells for veterinary regenerative medicine.
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374
Epidemiology of atopic dermatitis and other allergic skin diseases in
dogs and cats in Western Romania
Tiana SUICI, Gh. DARABUS, Narcisa MEDERLE, Mirela IMRE, C. SIRBU S. MORARIU
Facultatea de Medicină Veterinară Timișoara [emailprotected]
Abstract
Allergic skin diseases in dogs and cats are an emerging problem worldwide. In the past few years,
the number of cases has greatly increased and a rising interest in their treatment and diagnosis exists among
veterinarians. The aim of this study is to make a short description of the epidemiological situation of these
skin problems in the western part of Romania. The study was conducted during a 3-year period in a total of
8 clinics from three different counties in western Romania: two clinics from the Mehedinti County, three
clinics from Timis County and three clinics from Arad County. Reports were made regarding the number of
patients, which were presented at the clinics and were diagnosed with atopic dermatitis, flea allergy
dermatitis or other types of allergy (food allergy, contact allergy). This way we managed to obtain a
preliminary ratio of allergic affections in dogs and cats in the western part of Romania. The average value
for cats in all three counties was 10.74% of all cases presented at the clinics and 45.22% of the cases that
presented dermatological symptoms. The average value for dogs in all three counties was 5.94% of all cases
presented at the clinics and 42.49% of the cases that presented dermatological symptoms.
Keywords: allergy, dogs, epidemiology, western Romania
Introduction
Dogs, similar to humans, may develop a syndrome of spontaneous, inflammatory, pruritic
dermatitis with characteristics such as a young age of onset, characteristic distribution of lesions
and IgE sensitization to common environmental allergens generically called atopic dermatitis
(AD)(9). The main clinical features are skin lesions distributed around the mouth, eyes, ears, limbs
and ventral abdomen, pruritus and alopecia, erythema, conjunctivitis and recurrent otitis (8). The
clinical signs of AD and of all allergies in general can sometimes easily be mistaken for diseases
such as demodectic mange (6), Malassezia infections or skin diseases caused by Microsporum.
The diagnosis of canine AD is based either on the characteristic clinical features or on
results from various tests such as intradermal skin testing, IgE serology and even blood tests (BDT
or LTT tests) (5).
Epidemiological data help us understand the distribution, risk factors and causes of disease,
having a major influence in establishing a successful control and prevention protocol (12).
According to studies, AD and allergic skin diseases are one of the most common skin conditions
in dogs and cats with a prevalence of 3-15% in the general dog population and 3-58% of dogs
affected with skin diseases presented to veterinarians (8).
Materials and methods
The study was conducted over a three-year period (2014-2016) in a total of eight clinics
from three counties in western Romania: Mehedinti, Timis and Arad. The clinics taken into study
were as follows: Vet Point Vest, Vetagrica and Bioanima in Arad County, Ilivet and Negrostar in
Mehedinti County and Salvet, Dr. Ciolea Felician private practice and the Dermatology clinic of
FVM in Timisoara.
The information used in this paper comes from the consultation registers of the above-
mentioned clinics. A total number of 13.254 dogs and 4.708 cats were registered in these three
375
years in the eight clinics. The overall number of animals with a dermatological diagnosis was 2971
of which 1852 were dogs and 1119 cats. Results in detail are illustrated in the graphics and tables
that follow.
Table 1. Repartition of dogs presented in clinics from Timisoara, Arad and Mehedinti
Total cases of dogs
(no.)
Total of dermatological cases
(no.)
Total allergies
(no.)
Years Years Years
2014 2015 2016 2014 2015 2016 2014 2015 2016
4098 4514 4642 582 634 636 229 289 269
General total= 13254 General total= 1852 General total= 787
Table 2. Repartition of cats presented in clinics from Timisoara, Mehedinti and Arad
Total cases of cats
(no.)
Total of dermatological cases
(no.)
Total allergies
(no.)
Years Years Years
2014 2015 2016 2014 2015 2016 2014 2015 2016
1461 1553 1694 339 397 383 143 178 185
General total= 4708 General total= 1119 General total= 506
Table 3. Repartition of both cats and dogs in all three counties
Total cases
(no.)
Total of dermatological cases
(no.)
Total allergies
(no.)
2014 2015 2016 2014 2015 2016 2014 2015 2016
5559 6067 6336 921 1031 1019 372 467 254
General total= 17962 General total= 2971 General total= 1093
In all three counties the highest rate of dermatological diseases in dogs and cats was
recorded in 2015 when the rate was 16.99% of the total cases followed by 2014 with a rate of
16.57% and 2016 with a rate of 16.08% (figure 1).
376
Fig.1- Percentage of dermatological cases and AD or other allergic cases
Allergies were present in a higher rate in the year 2015 when the percentage of AD and
other allergic diseases was 7.70% of the total cases and 45.3% of the dermatological cases.
Gradually followed the years 2014 with a rate of 6.69% of the total cases and 40.4 % of the
dermatological cases and the year 2016 with a rate of 4.01% of the total respectively 24.9% of the
dermatological cases.
The overall percent of allergic skin conditions including AD was 6.1% during the period
2014-2016 out of a total number of 17,962 animals.
According to earlier research, the prevalence of AD in the canine population was estimated
at large to be 15% (1). More recently, scientific papers state estimates of 10 % (9). Unfortunately,
the true prevalence and incidence of AD in the general dog population is very difficult to establish
because of the slight unreliability of data caused mostly by the fact that mild cases are often
successfully managed with symptomatic therapy without a specific diagnosis and some clinical
manifestations of AD are difficult to recognize. Lund et al. (4) has conducted a study in the USA
on 31,484 dogs examined in 52 private practices and reached an estimate of 8.7% dogs diagnosed
with allergies. On the other hand, more recent studies also from the USA state a rate of 27%
incidence of AD among dogs and cats (10). Canadian studies (11) stated that AD stood for 12.7%
of the cases while in France, DeBoer’s (2) survey reports a 27% rate of 11,373 dogs.
Conclusions
In the western part of Romania, the total prevalence of AD and other allergic skin diseases
was highest in 2015 when the rate was 7.7%, AD or other allergic skin diseases representing 45.3%
of dermatological cases.
16.57 16.99 16.08
6.69 7.7
4.01
40.4
45.3
24.9
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
50.00
1 2 3
Total dermatological cases % Total allergy cases %
Allergies/Dermatological cases %
377
The overall rate of allergic skin diseases in Western Romania during the years 2014-2016
was 6.1% out of 17.962 animals.
Acknowledgements
This study is published under the frame of European Social Fund, Human Resources
Development Operational Programme 2007-2013, project No. POSDRU/159/1.5/S/132765 and
this research work was carried out with the support ofthe projectDezvoltarea infrastructurii de
cercetare, educaţie şi servicii în domeniile medicinei veterinare şi tehnologiilor inovative pentru
RO 05, cod SMIS-CSNR 266.
References
1. CHAMBERLAIN, K.,W., 1974-Atopic (allergic) dermatitis, Vet. Clin. N. Am. 4,29-39; 2. DEBOER, D., J., 1989-Survey of intradermal skin testing practices in NorthAmerica, J. Am. Vet. Med.
Assoc. 195, 1357-1363. 3. HILLIER A., GRIFFIN, C.E. 2001-The ACVD task force on canine atopic dermatitis (I): incidence and
prevalence; Veterinary Immunology and Immunopathology 81, 147-151; 4. LUND, E.M., ARMSTRONG, P.J., KIRK, C.A., KOLAR, L.M., KLAUSNER, J.S., 1999-Health status
and population characteristics of dogs and cats examined at private veterinary practices in the United States, J. Am. Vet. Med. Assoc. 214, 1336-134;
5. MARSELLA, R., (2013)-Atopic disease, In: Miller,W.H.J., Griffin, C.E., Campbell, K.L. (Eds.), Muller & Kirk’s Small Animal Dermatology, Seventh Ed. Elsevier Mosby, St. Louis,MO, USA, pp. 365–388
6. MEDERLE NARCISA, DARABUS GH., OPRESCU, I., MORARIU, S., ILIE, M., INDRE, D., MEDERLE, O., 2010- Diagnosis of canine demodicosis, Sci. Parasitol., 11 (1): 20-23
7. MILLER, W.H.J., GRIFFIN, C.E., CAMPBELL, K.L., 2013- Muller & Kirk’s Small Animal Dermatology, Seventh Ed. Elsevier Mosby
8. OLIVRY, T., SARIDOMICHELAKIS, M.,N. 2016 -An update on the treatment of canine atopic dermatitis, The Veterinary Journal 207, 29-37;
9. PUCHEU-HASTON C., M. 2016- Atopic dermatitis in the domestic dog, Clinics in dermatology, vol. 34, 2, 299-303
10. SCOTT, D.W., MILLER, W.H., GRIFFIN, C.E., 2001-Small animal dermatology, 6th Edition, W.B. Saunders, 574-601.
11. SCOTT, D.W., PARADIS, M., 1990- A survey of canine and feline skin disorders seen in a university practice: small animal clinic, University of Montreal, Saint-Hyacinthe, Quebec, Can. Vet. J. 31, 830-835;
12. TORRENCE, M.E., 1997- Understanding Epidemiology, Mosby Year Book, 1-17;
378
Generating bovine embryos through ICSI
Thomas KELLER, Simona MARC, Horia CERNESCU, Camelia TULCAN, Ioan HUȚU, Gabriel OTAVA, Ana-Maria RAȚIU, Georgiana UNGUREANU, Călin MIRCU
Banat University of Agricultural Sciences and Veterinary Medicine Timisoara, Faculty of Veterinary Medicine, Calea Aradului, no.119, 300645 Timişoara, jud. Timiş, Romania
[emailprotected]
Abstract
Through ICSI, competition between sperms and also sperm-oocyte interaction are avoided thus ICSI
proving reliable when sperm is not suitable for IVF. In bovine, the limiting step is represented by low rate
of sperm head decondensation subsequent ICSI. Intracytoplasmatic sperm injection allows avoiding many
critical moments that may occur during normal or in vitro fertilization. Oocytes were obtained from ovaries
from slaughtered cows. These were transported in 0.9% NaCl solution in isothermal bags at a temperature
of 25-30 ° C. The ovaries were brought from the slaughterhouse within 2 hours. Harvesting of the oocytes
was made through the aspiration method. After maturation, oocytes were fertilized using sperm that was
prepared using Percoll method and then treated with TritonX. The volume of the TritonX solution that
accompanies the sperma and which remains in the oocyte is extremely important given that by its action,
TritonX removes the acrosome, thus releasing a rich enzyme content and facilitating the dehydration of the
male pronucleus. Even though the number of 2 nucleus, 2 cells or 4 cells oocytes is inferior to the data found
in the literature, compared to the results achieved last year in the assisted reproduction laboratory within
CLC-HC Timisoara, it marks significant progress. At the 2 cells stage, there were several oocytes from group
1 (24.39% vs. 12.5%), while at the 4 cells stage there were 14.63% oocytes from group 1 and 25% group 2.
The use of TritonX solution for sperm treatment as well as shortening the duration of ICSI execution allowed
us to get encouraging results. The results obtained are inferior to those presented in the literature but are
far superior to those we obtained last year when the ICSI technique was assembled. Achieving the two- and
four-cell embryonic stages justifies us thinking that we are mastering the ICSI technique.
Keywords: bovine, embryos, ICSI, TritonX
Introduction
Intracytoplasmatic sperm injection allows avoiding many critical moments that may occur
during fertilization. Applying this technique involves sophisticated equipment as well as detailed
specialist knowledge. It is essential to have a good knowledge of working steps equal to the use of
certain specific reagents of the highest quality (Godja et al., 2016).
The ICSI technique in cattle was assembled in CLCHC Assisted Reproduction Laboratory
last year, and the results obtained determined us to continue its application. In the present paper,
we attempted to demonstrate the implications of using TritonX for sperm treatment as well as
reducing working times in order to shorten both the interval in which the sperm is in contact with
the slowing solution of their movements and the period of oocytes outside the incubator.
In cattle, intracytoplasmic sperm injection (ICSI) has low efficacy (Canel et al., 2014;
Zambrano et al., 2016). The content of acrosome may be considered responsible for this effect due
to the large number of hydrolytic enzymes released in oocytes.
In order to eliminate the acrosome and destabilize sperm membranes, researchers at the
University of Frontera (Zambrano et al., 2016) chose the use of Lizolecitin and Triton-X 100. The
rate of oocyte development was assessed along with pronuclear formation and embryo quality. The
use of Lizolecitin and Triton-X 100 (0.01%, 0.02%, 0.03%, and 0.04%) decreased the sperm
viability based on the dose used. At the same time, an improvement in the acrosome reaction for
all Lizolecitin and Triton-X 100 concentrations was observed, reaching already a concentration of
100% from 0.05% of both treatments.
379
A higher rate of cell division was observed in the intracytoplasmic injection of sperm treated
with Triton-X 100 (66%) and Lizolecitin (65%) compared to the untreated sperm group (51%). At
the same time, a significant increase in blastocyst formation was observed in the Lizolecitin treated
group (29%) compared to the control group (21%). There was no difference in the formation of
pronucleus and the number of embryos developed.
The researchers concluded that sperm treatment with Lizolecitin and Triton-X 100 improves
the rate of embryo development without affecting the quality of embryos produced by using this
technique.
Galli et al. (2003) studied the effect of various substances used for sperm pretreatment. In
the study, the sperm was activated with heparin, D-penicillamine, hypotaurine and epinephrine
before ICSI.
The effect of the use of dithiothreitol for pretreatment of spermatozoa or oocytes to be
injected was then studied. Activation of sperm with heparin in combination with epinephrine did
not improve the development of embryos following ICSI. Instead, the use of dithiothreitol for pre-
treatment of oocytes improved cell division and blastocyst formation in the case of inactivated
embryos and an acceleration of blastocyst development when embryos were activated. At the same
time, sperm pretreatment with dithiothreitol followed by ICSI showed a significant increase in
embryonic development rates over the first 7 days.
The study by Canel et al. (2014) aimed primarily at identifying how different treatments
applied to sem*n, such as sperm pretreatment with heparin and L-glutathione or sexing of sem*n,
can influence the development of embryos following intracytoplasmic sperm injection in cattle.
Cell division and blastocysts were assessed at 2 and 7 days after ICSI. The results of the
study show a significant increase in the rate of cell division and blastocysts in the pretreated batch
with heparin and L-glutathione group compared to the untreated group. The use of sperm sem*n
has also improved the cell division rate, with no major differences in blastocyst counts.
Research has also been carried out on the quality of oocytes and sem*n (Ohlweiler et al.,
2013). During the study, experiments were performed using better or worse quality oocytes and
sperm.
The blastocyst formation rate was significantly higher in the case of good quality oocytes
(23.3% versus 11.1% in the case of inferior quality oocytes), regardless of the characteristics of the
sem*n. At the same time, there was no major difference in the rate of blastocyst formation using
poor quality sem*n (regardless of oocyte quality). However, the use of good quality sem*n has
been shown to have a major influence on the blastocyst formation rate (25.7% versus 9.2% in the
case of poor quality oocytes).
The influence of oocytes and sperm quality on ICSI was subsequently evaluated. No major
differences were observed in the blastocyst formation rate irrespective of the quality of oocytes or
sem*n.
The conclusion of the researchers was that the use of intracytoplasmic sperm injection is an
effective way to achieve the in vitro generation of bovine embryos irrespective of the quality of
oocytes or sperm.
Materials and methods
Oocytes were obtained from ovaries from slaughtered cows. These were transported in 0.9%
NaCl solution in isothermal bags at a temperature of 25-30 ° C (Mayes, 2002). The ovaries were
brought from the Nojag slaughterhouse, which is 167 km (2h) from the Assisted Reproduction
Laboratory of the CLCHC and from Macea, Arad County, which is 80.5 km (1h 9min) from the
laboratory. For the harvesting of the oocytes, the aspiration method (Chung et al., 2000) was chosen
380
and cultivated for those of category I. After harvesting, COC, together with the follicular fluid,
were placed in a 50 ml conical tube for sedimentation. After 5 minutes of rest, they were sucked
from the bottom of the tube with the help of a pipette. This operation was repeated 4-5 times to
make sure all oocytes were sucked together with the cumulus cells.
The next step was to wash the COC by passing successively into 2 PBS plates. Thus, they
are prepared for maturing in 4-well plates. After being held in 400 μl of TCM 199 enriched with
10% ESC and coated with mineral oil to prevent oxygen action, for 22h for maturation in the
incubator, they were removed from the PBS-washed incubator and then denuded in 0.4 ml of 0.1%
hyaluronidase (Sigma) and washed in two steps of PBS.
The sperm strains stored in liquid nitrogen were thawed at 37 degrees for one minute
(Rahman, 2010). Sperm was prepared using Percoll method. Through the density gradient
(Percoll), dead, abnormal (low density) sperm and detritus (cell contamination) of ejacul*te
accumulate in the corresponding densities, while the density of the sperm (mobile, normal) crosses
the gradient and is found on the bottom of the tube. To eliminate all traces of gradient used, sperm
isolated by this method is washed in a culture medium. In order to centrifuge in the concentration
gradient, 90% and 45% Percoll solutions were prepared by dilution with Earl 1x solution. The
Percoll concentration gradient solution was kept on a water bath for 4 hours at 37 degrees before
use. 200 μl of sem*n from each bull was used. After bringing the samples to 37 ° C, they were
slowly added dropwise onto the walls of the centrifuge tube over the Percoll solution in a
concentration gradient (90%-45%) pre-heated for 4 hours at 37 ° C. Then the mixture was
centrifuged (Hettich 350R) for 20 minutes. After which the supernatant was removed and 2 ml of
Earl 1X solution was added. The mixture was hom*ogenized and analyzed for the determination of
seminal material parameters. To facilitate manipulation of the sperm and to immobilize it, the use
of Triton-X 100 (Sigma) was chosen. It is necessary to mix 50 μl of sperm suspension with an
equal volume of TCM 199 and 0.1% Triton-X 100. The mixture is centrifuged at 2000xG for 3
minutes.
After removing the cumulus cells, the oocytes were placed in a drop containing 5 μl of IVF-
TALP (in vitro fertilization-Tyrode's albumin pyruvate albumin). The sperm was transferred to 10
μl of the culture medium (Sp-TALP) containing 10 μl / ml heparin. ICSI was performed at a
magnification x200 microscope in drops of 30μl of TCM199 + BSA 3mg / ml medium coated with
silicone oil and kept at 37 ° C on the microscope warm plate. On the fertilization microplate were
placed two drops of TCM199 + BSA medium into which the oocytes were introduced and the drop
was covered with mineral oil. A drop containing spermatozoa treated with TritonX was placed on
the same microplate, from which a single sperma was extracted (Figure 1 A). The oocytes were
fixed in the micromanipulator pipette polar body at 6 o'clock, and the sperm was injected
perpendicularly (approximately at 3 o'clock) (Figure 1 B).
381
Fig.1. Sperm in micropipette (A) and injecting of sperm into oocyte (B)
After ICSI, the oocytes were transferred to the culture medium of TCM199 + 20% ESC
coated with silicone oil in 60x15 mm culture plates (Falcon, Fischer Scientific) and placed in the
incubator at 37 ° C, 100% humidity and 5% CO2. 18 hours after sperm injection, oocytes were
examined for the presence of the pronucleus. After 72h of ICSI, some oocytes were found to have
2 cells (Figure 2 A) and at 96 hours ICSI had 4 cells (Figure 2 B).
Fig.21. Oocytes with 2 cells (A) and 4 cells (B)
To highlight the division, oocyte staining with Hoechst 33342 (Thermofischer Scientific)
was chosen. After they were removed from the incubator they were washed in a PBS step after
which they were placed in Hoechst for 15 minutes in the incubator. After this period, they were
examined in an immunofluorescence microscope to highlight the cell division (Figure 3).
382
Fig.3. Before and after using the Hoechst staining, the presence of 4 cells is evidenced
Results and discussions Depending on the length of ICSI's own workload (from sperm contacting to injection into
oocytes), we divided the 73 oocytes injected into two groups (Table 1):
Group 1, in which we included the 41 oocytes injected in less than 7 minutes;
Group 2, containing 32 oocytes injected in more than 7 minutes.
We chose the 7 minute threshold taking into account both the recommendations in the
literature and our values (the shortest time was 4 minutes and 3 seconds and the longest 17 minutes
and 22 seconds), Values to which we calculated average and standard deviation. ICSI was
performed in TCM supplemented with 20% ESC, the oocytes remaining in this medium for the
first 24 hours. Subsequently, they were moved for another 24 hours in the maturation medium
containing cumulus cells and 48 hours after ICSI time they were again moved to the TCM +
ESC20% medium where they remained during the observation period. The 2N stage was also
highlighted by the 24-hour examination from ICSI, 2 cells (2C) at 72 hours from ICSI and 4-cell
(4C) at 96 hours from ICSI. The embryos remained in those stages for more than 24 hours, at which
time we found degenerative processes and lack of further development.
Table 1. Results obtained from ICSI on cattle oocytes
Group
Number
of
OV x
ICSI
Nonferti
lized
oocytes
Oocytes
with one
pronucleus
Oocytes
with two
pronucleus
Fertilized
oocytes that
have reached the
stage of 2 cells
Fertilized
oocytes
reaching the
4-cell stage
Group 1 41 1 6 18 10 6
Group 2 32 4 4 12 4 8
Group 1, including oocytes subjected to ICSI in less than 7 minutes, had only unfertilized
oocytes (2.45%). There were 6 oocytes with one pronucleus (14.63%) and 18 oocytes (43.90%)
with two pronucleus. Of these 18, 10 fertilized oocytes (24.39%) reached the stage of 2 cells and 6
oocytes (14.63%) - the stage of 4 cells (Figure 4).
383
The percentages of fertilized oocytes (as evidenced by the presence of the two pronucleus) as well
as those representing the 2 or 4 embryonic stages obtained at G1 are inferior to those presented in
the literature but clearly superior to those we personally obtained (Godja et al., 2016).
Gali (2003) reported only poor results subsequent ICSI as cell division and blastocist development,
despite bovine oocytes were activated and supposed to develop fertilization similarly with IVF.
Keskintepe and Brackett (2000) activated the oocyte at 30 minutes consequent sperm injection by
incubation with A23187 ionophore for 5 minutes. Bull sperm was previously capacitated by
incubation in a heparin-containing media. Authors reported 52,4% blastocyst division and 24,4%
for blastocyst development.
Fig. 2. Results from ICSI on cattle - Group 1
For group 1- containing oocytes at which ICSI occurred over a period of time longer than 7
minutes, there were 4 nonactive oocytes (12.5%) and 4 (12.5%) with a single pronucleus. Of the
12 oocytes (37.5%) who presented the stage of 2 pronucleus, 4 oocytes (12.5%) reached the stage
of 2 cells and 8 oocytes (25%) - the embryonic stage of 4 cells (Figure 5).
Figure 1 shows eloquently the differences between the two groups. Even though the number
of 2N, 2C or 4C oocytes is inferior to the data reported in the literature, compared to the results we
achieved last year (11) marks significant progress. It should be noted that at the 2C stage there were
several oocytes from G1 (24.39% vs. 12.5%), while at the 4C stage there were 14.63% oocytes
from G1 and 25% G2 (Figure 6).
We consider that the volume of the TritonX solution that accompanies the sperm and which
remains in the oocyte is extremely important given that by its action, TritonX removes the
acrosome, thus releasing a rich enzyme content and facilitating the dehydration of the male
pronucleus.
The results of the ICSI were also influenced by the running time of this work, and it is
desirable that it be as short as possible. The experience gained by the ICSI person makes it possible
to shorten this interval permanently, ensuring for oocytes spending as short a time as possible
outside the incubator.
384
Fig. 3. Results from ICSI on cattle - Group 2
Fig. 4. Results obtained from ICSI on cattle oocytes
We consider important to keep oocytes subjected to ICSI in the culture medium containing
cumulus cells, because this significantly improves oocyte metabolism by facilitating the exchange
of nutrients.
To grasp any statistical differences, we used the chi test, and a statistically significant
difference occurred at p ≤ 0.01 between G1 and G2, which is of particular interest when we look
at the results obtained with two or four cell embryos.
385
Stages C2 and C4 occurred 24 hours later than the accepted physiological time in bovine
embryogenesis, which discusses both the moment of potential oocyte activation and subsequent
survival.
The divergent opinions in the literature on whether or not the oocyte is activated following
the simple ICSI can support the underlying assumptions regarding the installation of the delayed
cell division and its closure after a short time as well as in the case of the 4C embryos the higher
success rate registered at G2 compared to G1. In other words, according to our results, the longer
duration of ICSI execution has enhanced cell division.
Conclusions
The use of TritonX solution for sperm treatment as well as shortening the duration of ICSI
execution allowed us to get encouraging results.
The results obtained are inferior to those presented in the literature but are far superior to
those we obtained last year when we assembled the ICSI technique.
Achieving the two- and four-cell embryonic stages justifies us thinking that we are mastering
the ICSI technique.
Aknowledgments
The research was carried in the IVF (In Vitro Fertilization) laboratory from the Horia
Cernescu Research Unit equipped through POSCCE 2669 program
References
1. CANEL, N. G., BEVACQUA, R. J., HIRIART M. I., SALAMONE, D. (2014) - IVF vs. ICSI: efficiency and “bull fertility effect” using sex sorted or non sorted sem*n in bovine, Animal Reproduction, 11, 3, 301-301.
2. CHUNG, J.T., KEEFER, C.L., DOWNEY, B.R. (2000). Activation of bovine oocytes following intracytoplasmic sperm injection (ICSI), Theriogenology, 53, 1273-1284
3. GALLI, C., VASSILIEV, I., LAGUTINA, I., GALLI, A., LAZZARI, G. (2003) Bovine embryo development following ICSI: effect of activation, sperm capacitation and pre-treatment with dithiothreitol, Theriogenology, 60, 8, 1467-1480.
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The behaviour pattern of several gastrointestinal nematode genera in
sheep and cattle from bethausen, Timis County C. SÎRBU, Gh. DARABUS, M. ILIE, Mirela IMRE, Tiana SUICI, S. MORARIU
Facultatea de Medicină Veterinară Timișoara Abstract
Parasitic infestations are one of the most important causes for animal disease and low productivity
worldwide. Gastrointestinal nematodes (i.e. Trichostrongylus, ) as well as trematodes (i.e. Fasciola spp.
Paramphistomum spp.), cestodes (i.e. Echinococcus spp.) and protozoa (i.e. Eimeria spp.) are all in the
category of most important parasitic diseases. Studies were conducted on cattle and sheep from Bethausen
village, Timis County. In cattle, the following genera were identified : Trichostrongylus, Cooperia and
Ostertagia while in sheep the Trichostrongylus, Ostertagia, Chabertia, Oesophagostomum and Haemonchus
genera were noticed. The present study was based on following the dynamics of the output of parasitic
elements from the April 2015 until March 2016. The best represented genus was Trichostrongylus both in
cattle and sheep with a prevalence of 40%, followed by Chabertia – 33%, Ostertagia – 30%, Haemonchus –
26% and Cooperia – 15%. The maximum EPG was achieved in October and the minimum EPG was achieved
in the months of January and February.
Introduction
Parasitic infestations are one of the main causes of animal sickness and low productivity
worldwide.
Parasitism is currently at a high point in farms and households on a global level, despite
many financial efforts directed towards control and prophylactic campaigns,. According to recent
research, the most significant damage is caused by pulmonary and gastrointestinal helminthosis.
However, it can be stated that these diseases ‘’don't kill the animal but destroy the farm”(5,6,23).
The fact that the parasites usually have a subclinical evolution (a phenomenon encountered
especially in temperate areas) in any temporary and/or permanent system of pasturing leads to a
decrease in the zootechnical performance.Several parasitic diseases can be mentioned in this
context: cryptosporidiosis, neosporosis, hydatidosis, fasciolosis, paramphistomosis and
trichostrongylosis. (4, 10, 11, 17, 18)
Thus, it causes considerable economical loss, due to the reduction of the growth rate, the
reduction of food conversion rate, the reduction of the milk and meat production leading to the
beginning of a subproductivity syndrome.The economic losses are due to expenses implied by
treatments although for some of them the possibility of vaccination exists (19, 20).
The economic influence of a subclinical evolution of the parasitosis can be rightly
appreciated only by taking into account all the elements, which are related to the pasture
contamination and to the receptivity of the animals.
The aim of this research was to investigate the parasitic spectrum, especially the
gastrointestinal nematodes, in a medium-sized village in Timis County, over a one-year period,
starting in April 2015 and ending in March 2016.
Materials and methods
The research took place in Bethausen Village , Timis County. Bethausen Village is situated
in the North-East of Timis County , on the right side of the Bega river, 26 km away from the town
of Lugoj and 20 km away from the town fa*get. Bethausen is situated in the centre and it is
387
surrounded by nearby villages as follows: Leucușești village in the East, Cutina Village in the
West, Cliciova and Nevrincea villages in the South and Cladova village in the North.
The village has a surface of 9027 hectares, of which 4764 ha are tillable, 1265 ha are
forests, 2066 ha pastures, 425 ha hays and orchards, 139 ha waters, 178 ha of roads and railways,
136 ha of country yards and buildings and 54 ha of non-productive fields. It is situated on both
parts of Bega (26).
Cattle and sheep are frequently found on the pasture, which has its own source of water (a
well with concrete gutters). The grass carpet had the following floristic composition: spontaneous
species of perennial Gramineae (Poa spp., Festuca spp., Dactylis spp., Bromus spp., Phleum
pratense) with small areas of leguminous plants (Trifolium repens, Lotus corniculatus) and other
plants from the spontaneous flora.
Between 50 of 150 gr of freshly eliminated faeces were collected or taken directly from
the rectum of the individuals .(4 cattle and 15 sheep)subjected to study. The samples were packed
in plastic bags and refrigerated until processing.
The samples were transported to the Parasitic Diseases laboratory of FVM Timisoara and
they were processed according to the following methods:
- Willis - to enhance de type of parasitism;
- McMaster - in order to find out the amount of parasites (EPG) in each individual
from the experiment;
- Euzeby - to quantify the pulmonary parasites in ruminants(2,3,6,7).
- Larvae cultures.
Only three genera were noticed in cattle: Ostertagia, Cooperia and Trichostrongylus. The
behaviour of the three-gastrointestinal nematodes was different according to season .
It can be seen that, during the spring months, the Cooperia genus was best represented in
April-39.62% and May-37.14%. The weakest prevalence was registered during winter months:
16.12% in January and 14.58% in February. The Ostertagia genus had the highest prevalence in
summer months.
The population peak was reached in July (51.45%) and the lowest numbers were recorded
in December (26.92%).
On the other hand, the Trichostrongylus genus seems to feel better in the winter months
when the rate is higher than 45%, peaking in December- 52.57%. The lowest values were observed
in summertime in July (13.60%).
In Sweden, Dimander (8) highlights the presence of 14 species of gastrointestinal
nematodes but the weight was held by 2 species: Ostertagia ostertagi and Cooperia
oncophora.Five genders of gastrointestinal nematodes were identified in sheep after reading the
larval cultures: Haemonchus, Ostertagia, Trichostrongylus, Chabertia şi Oesophagostomum.
It can easily be noticed that the Haemonchus genus has a relatively even distribution
throughout the study period except for the winter months when its prevalence dropped, reaching
values of zero in February. In addition, this genus has had the weakest infrapopulation
representation.
In an increasing order followed the Oesophagostomum genus which had a slightly
increasing trend throughout the entire period, especially during winter months with a peak (20%)
in February.
388
.
Fig.1 The behaviour of the Trichostrongylus genera
in the bovine host according to seasons
Fig. 2 The behaviour of gastrointestinal nematodes genera in the sheep
host according to seasons
The genus Chabertia occupied third place. It had a behaviour similar to the genus
Oesophagostomum, with a population peak in winter months when it reached 30%.
On the next position, we placed the genus Ostertagia that had its population peak in June-
July. However, the population suffered a drastic decline up to 0% in February. Probably this drop
of various Trichostrongylus infrapopulations is due to the hypobiosis phenomenon but also to aging
of individuals that form the population. In addition, as a consequence of the “spring-rise” or “peri-
parturient rise” phenomenon seen in the hypobiotic species (such as Haemonchus and Ostertagia),
389
the populations recover in spring, thus contributing to the pollution of pastures with parasitic
elements.
The most representative genus was Trichostrongylus, which had a relatively constant
prevalence, often situated around 40%, regardless of the season. It has to be pointed out that in the
case of species which do not use the hypobiosis phenomenon as a survival strategy, the
infrapopulations were larger. However, this population rise is in fact due to the lowering of the
individuals’ number of the species which use hypobiosis.
The results of such coproscopic investigations are subjected to a number of variables such
as: the moment of day in which the samples were collected (knowing that several species of
helmints lay more eggs in the morning and others do this in the evening), the age of helmints (those
who are elderly do not eliminate eggs), the abundance of female nematodes in the structure of the
infrapopulation, their fertility, the pathogeny of the species, the quality of the host’s immune
response, the grazing seasons, the host’s age, the consistency of faeces, etc. (9,15,22)
The study conducted by Odoi et all. (21) in Kenya has highlighted the presence of five
genera of gastrointestinal nematodes: Trichostrongylus (42,0%), Haemonchus (35,8%), Cooperia
(5,5%), Strongyloides (12,0%) and Oesophagostomum (4,7%). If in the case of Trichostrongylus
the results were similar to ours, in the case of Haemonchus we noticed only 1/3 of the population
reported in Kenya. Similar was the case of Oesophagostomum. In addition, we noted the presence
of Ostertagia and Chabertia and the lack of Strongyloides and Cooperia.
However, in a study conducted in Venezuela by Morales et all. (16) they reported the
presence of more gastrointestinal nematode genera, and the presence of more species:
Haemonchus, Trichostrongylus, Cooperia, Skrjabinema, Bunostomum, Oesophagostomum and
Trichocephalus.
In the American state of Nebraska, Colwell et all. (1) have observed that 99% of the
gastrointestinal nematodes presents in lambs had only two representatives: Ostertagia ostertagi
and Nematodirus helvetianus.
Theodoropoulos et al. (25) observed that in some arid regions of Greece, egg production
through faeces increased rapidly in the summer months (June to August). In India, Shing et al. (24)
had similar results to ours, with a low OPG in January-February and one that grew from July to
peak in September
The research conducted in Pakistan on several sheep flocks by Jan et all. (14) has
demonstrated a higher prevalence of parasitism with certain gastrointestinal nematodes in males
compared to females: Trichostrongylus spp. – 12,5%/0%, Haemonchus contortus – 13,5%/11,5%,
Chabertia ovina – 5,5%/1,5% while other nematodes were signaled only in females: Ostertagia
circumcincta – 7,5%, Oesophagostomum columbianum – 10,5%.
In Romania, in the sheep from the western and Northwestern parts of the Timis County,
Indre et all. (12) identified the following species of gastrointestinal nematodes, listed in a
decreasing order: Trichostrongylus – 37%, Chabertia – 33%, Ostertagia – 30%, Bunostomum –
27%, Haemonchus – 26%, respectively Cooperia – 15%. The linear distribution of these parasites
was as previously known with slight differences in what regards location: Trichostrongylus
colubriformis – 75% in the duodenum, 87,5% in the jejunum and ileum, respectively 12,5% in the
colon; Nematodirus filicollis – 42,85% in the duodenum and 85,71% in the jejunum and ileum or
in the case of Trichocephalus ovis – 11,11% in the jejunum and ileum, 66,66% in the colon,
respectively 88,88% in the cecum (13).
390
Conclusions
The coproscopic investigation made during cattle monitoring in Bethausen showed
parasitism with only three genera of gastrointestinal nematodes: Ostertagia, Trichostrongylus,
Chabertia.
Five parasitic genera were identified in sheep: Haemonchus, Ostertagia, Trichostrongylus,
Chabertia and Oesophagostomum.
In both cattle and sheep, the biggest output of parasitic elements was seen in October.
Acknowledgements
This study is published under the frame of European Social Fund, Human Resources
Development Operational Programme 2007-2013, project No. POSDRU/159/1.5/S/132765 and this
research work was carried out with the support ofthe projectDezvoltarea infrastructurii de cercetare,
educaţie şi servicii în domeniile medicinei veterinare şi tehnologiilor inovative pentru RO 05, cod
SMIS-CSNR 266.
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Preliminary research regarding the prevalence of digestive and
respiratory parasitosis in meat cattle from the Hârtibaci Valley,
Sibiu County
Radu NECHITI1, Gheorghe DĂRĂBUȘ2, Sorin MORARIU2 1 Sanitary and Veterinary Circ*mscription Nocrich, jud. Sibiu;
2 Faculty of Veterinary Medicine Timișoara [emailprotected]
Abstract
In Romania, the data regarding gastrointestinal and respiratory parasitism in cattle are scarce and
incomplete. This study was undertaken on Angus breed cattle from an intensive-type exploitation in
Nochrich, Sibiu County. The faeces samples were examined both through qualitative and quantitative
methods. The most widespread type of parasitism was the one caused by the ciliate Balantidium coli
(51.61%), followed in equal manner by Eimeria (32.25%) and Fasciola/Paramphistomum. The digestive
strongyles (22.58%) were less representative. These were followed in a decreasing order by the infestation
with Dictyocaulus viviparus (16.13%) and by Strongyloides spp. (12.90%).
Key words: Angus breed cattle, internal parasites, prevalence, Nocrich.
Introduction
In most cases, cattle are raised for milk, meat and skins but, more seldom, they can be used
to keep a good maintenance of pastures, various sporting activities (rodeo, corrida) and for
participation in contests in the agricultural field. Thus, they produce up to 90% of the total quantity
of milk consumed worldwide, 30% of the meat quantity and 90% of the skins used in the leather
industry (Acatincai, 2004).
The production forcing, along with the impact of dejections on the environment are
deficiencies in the management of cattle. The prejudice brought to the state of health (and implicitly
to production) by the presence of internal and external parasites should also be added. Although
cows can host more species of parasites in the digestive and respiratory tracts, only some of them
have a clinical or economic significance. We can list the species of the Ostertagia genus, located
in the abomasum, species of the Eimeria genus especially E. bovis and E. zuernii but also the
pulmonary nematode Dictyocaulus viviparus. Several species of trematodes like Fasciola hepatica
and those of the genus Paramphistomum to which you can add cryptosporidia, Neospora caninum
or Echinococcus granulosus as well as others can be mentioned (Darabus et al., 2011, Imre et al.,
2010, 2012, Morariu et al., 2011). Their transmission is achieved mostly through a digestive path
but it can also be realised transcutaneous, at pasture. In addition, the economic losses are owed to
the expenses of antiparasitic treatments though more alternatives that are efficient are available
like vaccines against some parasitic diseases (Morariu et al., 2005, 2010).
Bibliographic studies indicate a high incidence of parasitic diseases in cattle on an
international level, especially in the case of those raised in a free-range system, on grazing lands
(Darabus et al., 2006, Jager et al. 2005, Pilarczyk et al., 2009). This is the reason why the present
paper aims to establish the epidemiological situation regarding the internal parasitism in cattle
raised in an extensive system, especially of Angus breed cattle from the Hârtibaci Valley, Sibiu
County.
mailto:[emailprotected]
393
Materials and methods
The Hârtibaci Valley is found in the central-Eastern part of the Sibiu County and it is part
of the Hârtibaci Plateau. This area spreads on a surface of 237.515 ha, distributed as follows: 51%
in the Sibiu County, 35% in the Brașov County and 14% in the Mureș County (Figure 1).
Fig. 1-Etnographic areas of the Sibiu County
(after http://bjastrasibiu.ro/biblioteci-din-judet/)
During the year 2016, a number of Angus cattle were subjected to coproparasitic
examinations. They belonged to Karpaten Meat (KM) beef cattle exploitations in the Nochrich
area, Sibiu County (Table 1). Both quantitative and qualitative coproscopic methods were used
during examinations.
Results and discussions
From a total of 31 examined samples, using the above-mentioned methods, three were
negative (9.67%), nine were positive with a single parasite species - monoparasitism (29.03%) and
most of them presented polyparasitism, harbouring two or more species of parasites (61.29%).
The most widespread parasite was the ciliate Balantidium coli (51.61%), followed by
Eimeria (32.25%) and in equal measure, by Fasciola/Paramphistomum. Surprisingly, the digestive
strongyles were less representative (22.58%). These were followed, in a decreasing order, by
Dictyocaulus viviparus (16.13%) and by Strongyloides spp. (12.90%). The data are presented in
Figure 2.
The massive presence of B. coli denotes feeding deficiencies, which permitted an
excessive multiplication of the ciliate and its transmission in the cattle herd.
394
Eimeriosis is largely spread, both in Europe and on the other continents were cattle are
bred as well (Davoudi et al. 2011). The registered prevalence varied from country to country and
from region to region (Bangoura et al. 2012) from 8.25% in Iran (Heidari et al., 2014) and 93% in
Poland (Pilarczyk et al., 2009).
Table 1. The results obtained after the examination of faeces from Angus cattle
No. Owner Registrationno
. Sex Age (years) Observations
1. Karpaten Meat 27541 F 6 Digestive strongyles, Eimeria,
Balantidium
2. 31642 F 5 Eimeria, Balantidium
3. 18666 F 6 Eimeria, Balantidium
4. 8488 F 1 Negative
5. 9203 F 1,5 Digestive strongyles, Eimeria,
Balantidium
6. 8978 F 1,5 Negative
7. 2385 M 1,5 Digestive strongyles, Eimeria
8. 2389 M 8 months Digestive strongyles, Eimeria
9. 9306 F 8 months Digestive strongyles, Eimeria
10. 9256 M 2 Dictyocaulus
11. 9264 M 2 Dictyocaulus
12. 9250 M 2 Negative
13. 9254 M 2 Balantidium
14. 8021 F 10 months Balantidium
15. 8233 F 10 months Fasciola/Paramphistomum
16. 8078 F 10 months Fasciola/Paramphistomum
17. 8164 F 10 months Fasciola/Paramphistomum
18. 8369 F 4 Fasciola/Paramphistomum
19. 8301 F 4 Fasciola/Paramphistomum, Balantidium,
Strongyloides
20. 8256 F 4 Fasciola/Paramphistomum, Balantidium,
Strongyloides, Digestive strongyles,
Dictyocaulus
21. 8266 F 4 Fasciola/Paramphistomum, Balantidium,
Strongyloides
22. 1572 F 6 Fasciola/Paramphistomum
23. 4269 F 5 Fasciola/Paramphistomum, Eimeria
24. 9527 F 6 Fasciola/Paramphistomum, Digestive
strongyles, Dictyocaulus
25. 1637 F 6 Balantidium, Eimeria
26. 9526 F 7 Balantidium
27. 1634 F 7 Balantidium
28. 8104 F 2 Balantidium
29. 8138 F 2 Balantidium
30. 3400 F 4 Balantidium, Eimeria, Strongyloides,
Dictyocaulus
31. 3397 F 4 Balantidium
However, cryptosporidiosis has a different distribution and even in the neighbouring
areas, higher variations in values may be noticed: 5% in Sweden (Bjorkman et al., 2003) and 86.7%
in Tunis (Soltane et al., 2007).
Fasciolosis has affected 0.5% cattle in Turkey (Sariozkan and Yalcin, 2011) and 90.7%
of the Ethiopian herds (Behre et al., 2009) while Paramphistomum was present in 4.25% of the
395
cattle from Pakistan (Khan et al., 2008) and 53.4% of the Irish cattle population (Toolan et al.
2015).
Fig.2- The percentual repartition of types of parasites identified in Angus cattle
Dictyocaulus viviparus was diagnosed in 1.8% of the calves investigated in Costa Rica
(Jimenez et al. 2007) and 50% of the cattle investigated from a farm in Canada after the introduction
of carrier calves (Wapenaar et al., 2007).
Regarding the trichostrongyles, data are variable according to the implied genus and
according to the region. Thus, the prevalence ranges from 0.3% in Italy (Forbes et al., 2008) of
serum samples and 81.4% in Costa Rica (Jimenez et al., 2007).
For Romania, the data regarding gastrointestinal nematodes is abundant for sheep and
goats in the detriment of cattle. The research from the past few years has been addressed especially
to parasitic diseases in sheep. The most recent detailed research in this matter was conducted by
Avram (2003), who studied the situation of the internal parasitism in cattle from Satu-Mare County.
It was observed that the prevalence of parasitism with gastrointestinal nematodes ranged according
to region, having total values comprised between 38.6% and 66.2%. The most important genera
implied in the parasitic pathology of cattle from Satu-Mare County were in decreasing order:
Trichostrongylus, Cooperia, Ostertagia, Haemonchus and Nematodirus.
Conclusions
In the Karpaten Meat exploitation the infestation with various genera of parasites was
reported.
Parasites from three classes were identified: Protozoa, Trematoda and Nematoda.
The most widespread parasites were: Balantidium coli (51.61%), Eimeria and
Fasciola/Paramphistomum (32.25%), and less noticed were: Dictyocaulus viviparus (16.13%)
respectively Strongyloides spp (12.90%).
Acknowledgements
This study is published under the frame of European Social Fund, Human Resources
Development Operational Programme 2007-2013, project No. POSDRU/159/1.5/S/132765 and this
research work was carried out with the support ofthe projectDezvoltarea infrastructurii de cercetare,
educaţie şi servicii în domeniile medicinei veterinare şi tehnologiilor inovative pentru RO 05, cod
SMIS-CSNR 266.
10
20
30
40
50
60
Balantidium Fasc/Param Digestive strongyles Eimeria Strongyloides Dictyocaulus Negative
51.61
32.25
22.58
32.25
12.916.13
9.67
%
396
References
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ale bovinelor din județul Satu Mare. Teză de doctorat, USAMVB Timișoara. 3. Bangoura, B., Mundt, H.C., Schmaschke, R., Westphal, B., Daugschies, A., 2012 - Prevalence of Eimeria
bovis and Eimeria zuernii in German cattle herds and factors influencing oocyst excretion. Parasitol. Res.,
110, 875–881. 4. Behre, G., Berhane, K., Tadesse, G., 2009 – Prevalence and economic significance of fasciolosis in cattle
in Mekelle Area of Ethiopia.Trop. Anim. Health Prod., 41, 1503–1504. 5. Björkman, C., Svensson, C., Christensson, B., de Verdier, K. 2003 – Cryptosporidium parvum and Giardia
intestinalis in calf diarrhoea in Sweden. Acta Vet. Scand., 44, 145-152. 6. Davoudi, Y., Gharedaghi, Y., Nourmohammadzade, E., Eftekhari, Z.S., Safarmashaei, S., 2011 - Study
on prevalence rate of coccidiosis in diarrheic calves in East-Azarbaijan province. Adv. Environ. Biol., 5, 1563–1562.
7. Dărăbuș, Gh., Hotea, I., Oprescu, I., Morariu, S., Brudiu, I., Olariu, R.T., 2011 – Toxoplasmosis seroprevalence in cats and sheep from Western Romania. Rev. Méd. Vét., 162 (6): 316-320.
8. Dărăbuș, Gh., Oprescu, I., Morariu, S., Mederle, N., 2006 - Parazitologie și boli parazitare. Editura Mirton, Timișoara.
9. Forbes, A.B., Vercruysse, J., Charlier, J., 2008 - A survey of the exposure to Ostertagia ostertagi in dairy cow herds in Europe through the measurement of antibodies in milk samples from the bulk tank. Vet. Parasitol., 157, 1, 100-107.
10. Heidari, H., Sadeghi-Dehkordi, Z., Moayedi, R., Gharekhani, J., 2014 – Occurrence and diversity of Eimeria species in cattle in Hamedan province, Iran. Vet. Med., 59, 6, 271–275.
11. Imre, K., Dărăbuş, Gh., Mederle, N., Oprescu, I., Morariu, S., Ilie, M., Hotea, I., Imre, M., Indre, D., Balint, A., Sorescu, D., 2010 - Intraspecific characterization of some Cryptosporidium parvum isolates from calves and lambs in Western Romania using molecular techniques. Sci. Parasitol., 11 (1), 47-50.
12. Imre, K., Morariu, S., Ilie, M.S., Imre, M., Ferrari, N., Genchi, C., Dărăbuş, Gh., 2012 – Serological survey of Neospora caninum infection in cattle herds from western Romania. J. Parasitol., 98 (3): 683-685.
13. Jäger, M., Gauly, M., Bauer, C., Failing, K., Erhardt, G., Zahner, H., 2005 - Endoparasites in calves of beef cattle herds: management systems dependent and genetic influences. Vet. Parasitol., 31, 3, 173-191.
14. Jiménez, A.E., Montenegro, V.M., Hernández, J., Dolz, G., Maranda, L., Galindo, J., Epe, C., Schnieder, T., 2007 - Dynamics of infections with gastrointestinal parasites and Dictyocaulus viviparus in dairy and beef cattle from Costa Rica. Vet. Parasitol., 148, 3-4, 262–271.
15. Khan, U.J., Tanveer, A., Maqbool, A., Masood, S., 2008 – Epidemiological studies of paramphistomosis in cattle. Vet. Arhiv, 78, 3, 243-251.
16. Morariu, S., Bart, J.M., Cosoroabă, I., Morariu, F., Ilie, M., Dărăbuş, Gh., 2011 - Prevalence of cystic echinococcosis in sheep, cattle and swine from the western Romania. Sci. Parasitol., 12 (1): 47-51
17. Morariu, S., Lightowlers, M.W., Cosoroabă, I., Dărăbuş, Gh., Bart, J.M., Ilie, M., Morariu, F., Morar, D., Oprescu, I., Mederle, N., Imre, K., Belean, M., 2010 - Utilization of EG95 vaccine for sheep immunization against cystic echinococcosis in Romania. Sci. Parasitol., 11 (1): 29-34.
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97-104. 19. Pilarczyk, B., Balicka-Ramisz, A., Kozak, W., Ramisz, A., 2009 - Occurrence of endoparasites in heifers
imported to Poland from the Netherlands. Arch. Tierzucht, 52, 265–271. 20. Sariözkan, S., Yalçin, C., 2011 - Estimating the total cost of bovine fasciolosis in Turkey. Ann. Trop. Med.
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Cryptosporiidae) in calves: results of a longitudinal study in a dairy farm in Sfax, Tunisia. Parasite, 14, 4, 309–312.
22. Toolan, D.P., Mitchell, G., Searle, K., Sheehan, M., Skuce, P.J., Zadoks, R.N., 2015 - Bovine and ovine rumen fluke in Ireland - prevalence, risk factors and species identity based on passive veterinary surveillance and abattoir findings. Vet. Parasitol., 212, 3-4, 168-174.
23. Wapenaar, W., Barkema, H.W., Eysker, M., O'Handley, R.M., 2010 - An outbreak of dictyocaulosis in lactating cows on a dairy farm. JAVMA, 231, 11, 1715-1718.
24. *** - http://bjastrasibiu.ro/biblioteci-din-judet/
397
Research on metabolic status in periparturient cows
1Sorin D. SORESCU, 1Carmen IONIȚĂ, 2Alice GRIGORE, 1Emilia BALINT, 1Cosmin ȘONEA 1Ana Maria GOANȚĂ, 1Roxana ȚÎMPĂU, Lucian IONIȚĂ
1FMVB Facultatea de Medicină Veterinară București, 105 Splaiul Independenței, Bucharest 2National Institute for Chemical andPharmaceutical Research and Development (ICCF),
112 Calea Vitan, Bucharest, [emailprotected]; [emailprotected]; [emailprotected]
[emailprotected]; [emailprotected]; [emailprotected]
Abstract
In the experiment, hematological and blood biochemical parameters were determined in a batch of
5 cows in the last week of gestation (Group 1) and 5 cows in the first week after calving (Group 2). Cows
are clinically healthy and come from a farm where the milk production per fed animal is about 30 liters/day,
cows being milked 3 times per day. Hematologic parameters were found within physiological limits, but in
both groups the monocytes were found to be low, and in group 1, mild lymphopenia was detected.
Investigated blood biochemical parameters allowed to assert that in cows in the last week of gestation,
bilirubin was found to be significantly increased when recently-bred cows were within normal limits. In both
lots, LDH was found to be significantly increased. Metabolic status also determined the protein fractions by
means of electrophoresis: 10 samples were analyzed (Group 3 consisting of 5 cows in the last week of
gestation and Group 4 consisting of 5 cows in the first week after calving). The values of the protein fractions
were within the physiological limits and the Albumin/Globulin Ratio was found within physiological limits
in group 3 and lower in group 4, which confirms gamma globulin reactivation immediately after calving.
Key words: metabolism, parturition, protein fractions, immunoglobulins.
Introduction
It is known that a lot of changes occur in the dairy cow during the transition period (21
days before parturition and 21 days after parturition). In this study we tried to obtain a more detailed
table of the matabolic status of the periparturient cows closer to the moment of parturition (7 days
before parturion and 7 days after parturition) (5, 9). Dairy cattle, like many other species, often
consume less feed in the week prior to parturition (Grummer et al., 2004), and it can take up to a
week post-calving before dry matter intake (DMI) exceeds what the cow was consuming in late
gestation (6). The fatty liver present at one day after calving is negatively corelated whith feed
intake one day prepartum (3).
Metabolic disorders are a key problem in the transition period of dairy cows and often
appear before the onset of further health problems. Problems derive from the difficulty of the
animals to adapt to large variations and disturbances occurring outside and inside the organism. (4)
Oxidative stress is also known to be an important factor of the metabolic dysfunctions during this
period. (Miller et al., 1993; Sordillo and Aitken, 2009). A lack of success in solving these issues
may be due to predominant approaches in farm management and agricultural science. Instead, a
successful adaptation of animals to their living conditions should be seen as an important end in
itself. Both farm management and agricultural sciences should support animals in their ability to
cope with nutritional and metabolic challenges by employing a functional and result driven
approach (9). Techniques of modern hematology and biochemistry promise to further our
understanding of the mechanisms of metabolic adaptation during the peripartal period, and to
quantify the effects of nutrition and environment during pre-and postpartum periods on hepatic
glucose and lipid metabolism (1,2,7).
Another important aspect of the blood and its constituents is the fact that is very dependable
on the medium (temperature, way of collecting, stresss of collecting the blood) for evaluating
mailto:[emailprotected]
mailto:[emailprotected]
mailto:[emailprotected]
mailto:[emailprotected]
mailto:[emailprotected]
398
physiological changes in the physical and health status of an animal (Egbe-Nwiyi et al., 2000;
Žvorc et al., 2006; Njidda et al., 2014).
Materials and methods
In this paper we aim to achieve a metabolic monitoring of the main biomacromolecules
(proteins, lipids, carbohydrates) and enzyme and mineral status to prevent possible dismetabolites,
which once detected could be rectified so that even the worst period of gestation , the transition
period provides optimal comfort for completing gestation and obtaining healthy newborns.
To complete the research, we chose as location a cow farm near the capital, with a tradition
of raising dairy cows. At present, the farm hosts 574 cows, of which 309 cows, 65 heifers, 172
calves over 6 weeks, 18 calves above 6 weeks and 10 other categories. Cows are kept in free
standing on straw bedding. As experimental protocol, we made 4 groups, each one consisting of 5
cows, as follows:
Group 1: 5 cows in the last week of gestation and Group 2: 5 cows in the first week after calving,
from which we collected blood samples in order to compare the biochemical and haematological
blood parameters between the 2 groups;
Group 3 and group 4 with a similar consitency, but in this case, we collected blood samples for
determination of the protein fractions using a technique of electrophoresis; all paraclinical
examinations were performed in our discipline laboratory.
Results and discussion
As presenting in the table below, the hematological exam in cows during the gestation
week reveal a monocytopenia present in all cows from group A and also a lymphocitopenia present
at four of the cows from this group. The other parameters were found within physiological limits.
Table 1. The Results of the Hematological Exam at Cows in the Last Week of Gestation
(Group1) PARAMETER U/M Physiolog.
limits
Cow
71055
Cow
27829
Cow
53538
Cow
65317
Cow
8126
WBC 10-9/mm3 4-12 7,72 6,60 8,75 13,22 8,82
LYM 10-9/mm3 2,5-7,5 2,56 3,64 3,76 5,61 3,30
MON 10-9/mm3 0-1 0,08 0,06 0 0,12 0,13
NEU 10-9/mm3 0,6-7,6 4,88 2,01 4,75 7,18 5,19
EOS 10-9/mm3 0,1-1 0,19 0,10 0,16 0,30 0,20
BAS 10-9/mm3 0-0,5 0 0 0,01 0,01 0,01
LYM % 45-75 33,2 55,1 42,9 42,4 37,4
MON % 2-7 1,0 0,9 0,9 0,9 1,5
NEU % 15-65 63,3 42,5 54,3 54,3 58,8
EOS % 1-8 2,5 1,5 1,9 2,3 2,2
BAS % 0-3 0,1 0 0,1 0,1 0,1
RBC 10-12/mm3 5-10 6,65 6,96 7,34 8,18 7,29
HGB g/dl 8-15 10,8 9,6 11,3 11,9 11,5
HCT % 24-46 33,43 29,44 33,51 37,55 34,59
MCV fl 40-60 50 42 46 46 48
MCH pg 11-17 16,2 13,8 15,3 14,5 15,7
MCHC g/dl 30-36 32,2 32,6 33,6 31,7 33,1
PLT 10-9/mm3 100-800 250 362 392 340 425
399
Monocytopenia is known to appear as a result of aplastic anemy, pancytopenia and also
after using medication like: prednisolon, alprazolam, triazolam, but in this case we will strictly
corelate it with the advanced stage of getation. Monocytes are the largest cells in the blood; are
released into the blood and after a short while in circulation, migrate into different tissues,
incidentally or specifically, in response to various chemotactic factors. In tissues, in response to
different soluble factors, they differentiate into tissue macrophages with characteristic
morphological and functional qualities, a process that has been called "activation" and which is
reversible ("deactivation"). The cells of the phagocytic mononuclear system are very primitive
phylogenetic, and no animal can live without them. They perform a wide variety of important
functions in the body, including removal of foreign particles and senescent cells, dead or altered,
regulation of other cell functions, processing and presentation of antigens in immune reactions,
participation in various inflammatory reactions, destruction of bacteria and tumor cells.
Table 2. The Results of the Hematological Exam at cows in the first week after calving
(Group 2)
PARAMETER U/M Physiolog.
limits
78178 80501 23747 8096 8181
WBC 10-9/mm3 4-12 10,43 8,44 8,72 9,54 9,43
LYM 10-9/mm3 2,5-7,5 4,59 3,09 2,92 5,42 5,37
MON 10-9/mm3 0-1 0,11 0,04 0,85 0,09 0,16
NEU 10-9/mm3 0,6-7,6 5,41 4,02 4,64 3,70 3,69
EOS 10-9/mm3 0,1-1 0,32 0,47 0,30 0,32 0,21
BAS 10-9/mm3 0-0,5 0,01 0,01 0,01 0,01 0,01
LYM % 45-75 44 46,1 33,5 56,8 57,0
MON % 2-7 1,0 0,5 9,7 0,9 1,7
NEU % 15-65 51,9 47,7 53,2 38,7 39,1
EOS % 1-8 3,1 5,6 3,5 3,4 2,2
BAS % 0-3 0,1 0,1 0,1 0,1 0,1
RBC 10-12/mm3 5-10 7,3 5,51 6,39 6,65 8,00
HGB g/dl 8-15 11,2 9,0 10,7 9,7 11,3
HCT % 24-46 35,65 28,36 32,14 30,91 34,57
MCV fl 40-60 49 51 50 46 43
MCH pg 11-17 15,4 16,4 16,8 14,6 14,2
MCHC g/dl 30-36 31,5 31,9 33,3 31,4 32,7
PLT 10-9/mm3 100-800 242 257 294 379 297
Regarding Group 2, the results of hematological exam are very similar to those from group
1, the values are very little semnificative modified according to the physiological vallues.
At the biochemical examination of the blood, it was noticed an increasing of Lactat
Dehydrogenase (LDH) and Total Bilirubin (T-bil), which is directly corelated with the fiziological
status of the cows from Group 1. There are also variations of he other parameters: T-Cho,
Creatinine and Uric acid.
400
Tabel 3. The Results of the Biochemical Exam at Cows in the Last Week of Gestation
(Group 1) Parameter U/M Physiolog.
limits
71055 27829 53538 65317 8126
T- Pro g/dl 5,8-8,5 6,9 6,5 7,1 6,8 6,3
Albumin g/dl 2,5-3,7 2,9 2,7 2,7 3,3 3,5
Globulin g/dl 3,3-4,8 4,0 3,8 4,4 3,5 2,8
BUN mg/dl 10-25 10 11 9 11 13
UA mg/dl 1,0-2,1 0,9 0,8 1,0 1,1 0,9
Cre mg/dl 0,4-1,0 1,4 1,0 1,0 1,2 1,1
T-Cho mg/dl 70-280 59 50 75 85 71
GOT IU/l 78-132 88 89 68 91 92
LDH IU/l 692 1445 1348 1422 1654 1637
T-Bil mg/dl 0-0,3 1,0 0,6 0,4 0,7 0,5
GPT IU/l 0-82 8 7 9 12 7
ALP IU/l 0-80 118 113 110 135 69
Table 4. The Results of the Biochemical Exam at cows in the first week after calving
(Group 2) Parameter U/M Physiolog.
limits
78178 80501 23747 8096 8181
T- Pro g/dl 5,8-8,5 6,4 6,6 6,2 7,1 5,4
Albumin g/dl 2,5-3,7 3,2 3,4 3,3 3,3 3,2
Globulin g/dl 3,3-4,8 3,2 3,2 2,9 3,8 2,2
BUN mg/dl 10-25 9 13 13 7 9
UA mg/dl 1,0-2,1 0,9 0,7 0,7 0,8 0,7
Crea mg/dl 0,4-1,0 1,1 1,2 1,5 1,0 1,2
T-Cho mg/dl 70-280 98 66 98 92 77
GOT IU/l 78-132 113 51 48 42 76
LDH IU/l 692-1445 2343 1973 1561 1588 2256
T-Bil mg/dl 0-0,3 0,5 0,3 0,4 0,3 0,3
GPT IU/l 0-82 10 7 7 7 10
At the biochemical examination of the blood collected from group B, it is noticed that the
values of LDH and T-Bil continue to be increased and also, Creatinine reveals an increasing. Uric
Acid seems to be decreased at all the cows from this group.
Table 5. Determination of protein fractions by electrophoresis in Group 3 Fraction U/M Physiolog.
limits
8181 8096 23746 78178 80501
Total Protein g/dl 5,8-8,5 5,40 7,10 6,20 6,40 6,60
Albumin g/dl 1,3-2,47 1,81 1,47 1,51 1,87 1,84
α1 g/dl 0,19-0,78 0,27 0,30 0,37 0,31 0,33
α2 g/dl 0,19-0,78 0,50 0.63 0,63 0,67 0,62
β1 g/dl 0,32-0,84 0,80 0,95 0,79 0,93 0,77
β2 gd/l 0,32-0,84 0,49 0,61 0,61 0,53 0,47
γ g/dl 1,75-2,72 1,54 3,14 2,30 2,09 2,56
Alb/Glob / 0,45-1,31 0,50 0,26 0,32 0,41 0,39
401
Determination of protein fractions reveals insignificant changes according to the
physiological limits for the cows in the last week of gestation. The decrease Albumin/Globulin
ratio is associated with the physiological status of the cows.
Fig. 1 Distribution of Albumin/Globulin Ratio in Group 3
Table 6. Determination of protein fractions by electrophoresis in Group 4 Fraction U/M Physiolog.
limits
27189 53538 65317 71055 8126
Total Protein g/dl 5,8-8,5 6,50 7,10 6,80 6,90 6,30
Albumin g/dl 1,3-2,47 1,93 2,08 2,20 2,62 2,76
α1 g/dl 0,19-0,78 0,42 0,35 0,10 0,41 0,30
α2 g/dl 0,19-0,78 0,60 0,79 0,78 0,49 0,40
β1 g/dl 0,32-0,84 1,00 0,75 0,97 0,69 0,64
β2 gd/l 0,32-0,84 0,34 0,57 0,76 0,52 0.35
γ g/dl 1,75-2,72 2,21 2,57 1,99 2,17 1,86
Alb/Glob / 0,45-1,31 0,42 0,41 0,48 0,61 0,78
At one week after calving, the results of the elecrophoresis reveals very unsemnificative
changes according to the physiological limits, as follows: 2 of the cows from this group had a
decreased Albumin/Globulin Ratio and other two cows had an increased value of the albumin
vallue. This fact might be associated with the feed intake and also with some interferences.
Fig. 2 Distribution of Albumin/Globulin Ratio in Group 4
20%
80%
Albumin/Globulin Ratio in Group 3
Normal
Modified
40%
60%
Albumin/Globulin Ratio in Group 4
Normal
Modified
402
Conclusions
Clnically, all the cows taken in our study are clinically healthy, but at a routine blood exam
reveals a lot of changes in their metabolic status, as follows: monocytopenia, lymphocitopenia and
increased values of Total Bilirubin and Lactat Dehidrogenasys in the first two groups of cows.
There are no major diferences between the cows one week prior parturition and the cows
after parturition, due to the period taken into account for this study, but regarding albumin/ globulin
ratio it was noticed that in the cows before parturition is more significant modified than in the cows
after parturition, which confirms gamma globulin reactivation immediately after calving.
By correlating the results obtained, it can be argued that routine explorations are an
important tool in the diagnostic of some problems with the management of the farm and also, of
the animals like: alimentary disorders, deficiency of food intake, oxidative stress.
References
1. Calamari L., Ferrari Annarita, Minuti A., Trevisi E. (2016), Assessment of the main plasma parameters included in a metabolic profile of dairy cow based on Fourier Transform mid-infrared spectroscopy: preliminary result, BMC Vet Res. 2016; 12:
2. Fischbach F. (2009), Manual of Laboratory and Diagnostic Tests Lippincott, Williams & Wilkins, USA, 8 Ed., 2009, 199-210
3. Grummer R.(1993), Etiology of Lipid-Related Metabolic Disorders in Periparturient Dairy Cow, J. Dairy Sci 76:3882-3896
4. Ioniţă L. (2008). Patologie şi clinică medicală veterinară. Vol I, Editura Sitech, Bucureşti 5. James K.Drackley, Thomas R.Overton, G. Neil Douglas (2010). Adaptations of Glucose and Long-
Chain Fatty Acid Metabolism in Liver of Dairy Cows during the Periparturient Period, Veterinary Immunology and Immunopathology
6. Lorraine M. Sordillo*, G. A. Contreras and Stacey L. Aitken (2009). Metabolic factors affecting the inflammatory response of periparturient dairy cows, Department of Large Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824, USA
7. Roland L., Drillich M, Iwersen M., (2014), Hematology as a diagnostic tool in bovine medicine,
Journal of Veterinary Diagnostic Investigation, Vol. 26(5) 592 –59 8. Sundrum A., Clive J. C. Phillips (2009), Metabolic Disorders in the Transition Period Indicate that
the Dairy Cows’ Ability to Adapt is Overstressed, BMC Vet Res. 2009 9. Weary D., Keyserlingk G., Rushen J., (2009), The welfare of dairy cattle - Key concepts and the role
of science, J. Dairy Sci. 92 :4101–4111, American Dairy Science Association.
403
New Zealand Crossbred male rabbitproduction performance fed with
fructooligosacharide prebiotic isolated from banana peel
Suraya KAFFI SYAHPURA1), Kusmajadi SURADI2), Husmy YURMIATI2), Diding LATIFUDIN2) 1) Department of Animal Husbandry, Polytechnic Lampung,
Jl. Soekarno – Hatta no. 10, Rajabasa, Bandar Lampung 35144, Indonesia 2) Faculty of Animal Husbandry, Universitas Padjadjaran,
Jl. Raya Bandung – Sumedang Km. 21, Jatinangor - Sumedang 45363, Indonesia Email : [emailprotected]
Abstract The study was conducted to determine the production performance (growth performance and
carcass production) of New Zealand White Crossbred (NZWC) male rabbits given FOS
(fructooligosaccharide) from the isolation of Ambon banana peel (Musa paradisiaca var. Sapientum (L.)
Kunt. Rabbit age of eight weeks, body weight average 496.12 ± 10.61 g, placed in individual cage, divided
randomly into five treatments, and each treatment was repeated five times, each repetition consisted of two
rabbits The treatment used was P0 (without FOS), P1 (FOS fed for 7 days), P2 (FOS fed for 14 days), P3
(FOS fed for 21 days), and P4 (FOS fed for 28 days). FOS treatment is 3% concentration, 2 mg / kg of body
weight given orally, after 7 days adaptation. Drinking water is given ad libitum. The feed used is commercial
feed, given two times a day (in the morning and afternoon). The data were analyzed with analysis of
variance. The similarity of variance was tested using Barlett test and orthogonal polynomial test. Results
indicated that there were interaction between without FOS fed and FOS fed, and has effect on the growth
performance (feed intake, daily gain, slaughter weight, and feed conversion) are quadratic at 1% level, and
carcass production (carcass weight, carcass percentage, meat weight, bone weight, and fat weight) are
linear at 1% level.
Keywords: banana peel, fructooligosaccharide, performance, rabbit
Introduction
The New Zealand White Crossbred (NZWC) rabbit has been selectively developed in the
meat production unit, primarily for its propagation properties, namely prolification, performance,
rapid growth and rapid sexual development, size and uniform litter size and mothering ability and
high milk production (Lukefahr, 1983, Lebas et.al., 1996; Sarwono, 2002; Raharjo, 2005). The
demand for rabbit meat has been increase because of the various preparations that have been
circulating in the community. Efforts that can be made to meet the demand for rabbit meat is to
increase livestock populations with the consequent increase in land and feed requirements.
Moreover, it can be done by increasing the individual production capability. Efforts to increase
production capability can be achieved such as growth manipulation, by improving the quality of
rabbit rations. The given rations should meet the needs of the rabbit and contain balanced nutrition
that can support towards the achievement of optimal growth so achieves optimal production. One
of the efforts to achieve the above objectives is giving FOS (Fructooligosaccharide) from the
banana peel of industrial waste that still has nutritional value that serves as a prebiotic. Based on a
number of studies revealed contains vitamin C, vitamin B, calcium, protein, carbohydrates and
fiber are good for the body; so the banana peel still have the benefit to be used as animal feed, and
the benefits will be better through the extraction and isolation that can produce FOS
(Fructooligosaccharide), according patented number P00201406459 of the method of making FOS
from banana peel (Musita, 2008, Kaffi, et al. 2014). FOS is an alternative solution for control of
colibasilosis as an antimicrobial substance that has the ability to eliminate pathogenic bacteria in
the digestive tract and as immunostimulant (to stimulate the immune system). The fructans
404
contained in FOS produced by hydrolysis of chicory plant inulin have been widely reported, but
the production of FOS derived from banana peel waste has not been widely published.
Fruktooligosaccharide (FOS) in banana peel is potential to be developed as a prebiotic that
can reduce the number of pathogenic bacteria, and improve the morphology of the intestine (colon)
and also the thickness of the mucus layer. Fruktooligosaccharide (FOS) has an affinity to binding
the bacterial cells in intestinal epithelium. Bacteria that have binding to FOS can not stick and
colonize the intestinal wall, and finally carried out through the feces (Murniasih, 2010), besides
FOS banana peel also contains fructan sugar β (2-1) which is a prebiotic oligosaccharide. Prebiotics
are material /components that can be useful for the development of microflora in the intestines, to
be fermented by lactic acid bacteria especially Bifidobacteria and Lactobacillus; will also produce
short-chain fatty acids which can be used in the body as a source of energy. The implementation
to meet the meat quantity and quality, if problems to achieve the current economic conditions, it
can be done by diversifying the supply of animal protein, as the target of sustainable development
goals (SDGs), namely the achievement of people's welfare. One alternative to meet the needs of
meat/animal protein is from rabbit meat, because of prolific livestock, rabbits also produce good
quality and quantity of meat. On the other side, needs special attention in the maintenance, because
of the high mortality, so the utilization of prebiotic FOS from banana peel is expected to increase
the intestinal microflora and rabbit immune system, which is expected to increase the growth and
better meat quantity and quality. Many advantages derived from eating rabbit meat, namely high
protein content and low cholesterol, so the rabbit meat can be promoted as healthy meat, and also
the skin and the manure still have economic value (Yurmiati, 2006). The development of rabbit
meat as a provider to date still encountered many obstacles because the rabbit meat has not been
popular by some people making difficulties in rabbit meat marketing.
Materials and methods
Fifty male NZWC rabbits age eight weeks with the average body weight 496.12 ± 10.61 g
divided randomly into five treatments. Each treatment consists of five replications and two rabbits
in each repetition. The treatment are (P0) as the control treatment without giving FOS
(Fructooligosaccharide), (P1) given FOS for 7 (seven) days, (P2) given FOS for 14 days, (P3) given
FOS for 21 days, and (P4) which is giving FOS for 28 days. The FOS is derived from the isolation
of Ambon banana peel (Musa paradisiaca var. Sapientum (L.) Kunt derived from banana chips
industry using FOS isolation method from banana skin, patented number P00201406459 from
Indonesian Law and Human Rights Department. 100 kg of banana peel which had been chipped in
2-3 cm sizes, soaked in 30 L ethanol 70% for 14 days. During the daily immersion, stirring for 10
minutes, and the filtrate was filtered using a filter cloth and evaporated with a vacuum evaporator
up to 1 L. The concentrated filtrate is then extracted with ethyl acetate (EtOAc) to obtain a water
fraction and EtOAc. The water fraction was evaporated to dryness and then incorporated in a LH-
20 diaion chromatographic column then eluted with 3 liter H2O and 3 liter MeOH-H2O (3:7), also
with 3 liter MeOH-H2O (7: 3), and 3 liter MeOH, respectively. Each fraction was qualitatively
tested with TLC to determine the presence of FOS compounds. The fraction containing FOS, then
further purified by using purification techniques such as chromatography column, Preparative Thin
Layer Chromatography (PTLC), or crystallization. The FOS compounds obtained were then
analyzed by spectroscopy. The presence of compounds present in the solution can be qualitatively
and quantitatively identified with HPLC instruments based on standard solutions, to use as FOS
standard solution. The peak emerging from the solutions is a specific retention time of each
compound. Each fraction obtained was tested by TLC (Thin Layer Chromatography) method by
dripping on plate. The plate was developed with a combination of methanol-water solvent to get
405
the spot. Tests were also performed by comparing the retention time of standard FOS compounds
by using high performance liquid chromatography (HPLC) method using ODS (C-18) columns
and mobile phase used Methanol:water with ratio of 20:80. The detector used is UV-vis with a
range of 460-600 nm, with a 20 μm injection volume over 10-20 minutes (Kaffi, et al, 2014).
The data of FOS was performed after the rabbit was kept for 7 days for environmental
adaptation, giving orally according to each treatment; once daily in the morning between 07- 08
a.m with 3% concentration,doses of 2 mg / kg body weight (Prata and Mussato, 2010), , and
drinking water is given ad libitum. The rabbit is placed in an individual rabbit cage (50 x 40 x 35
cm) with eating and drinking apparatus of 50 enclosures, which are kept for eight weeks and also
was given the Indofeed commercial rabbit feed. The second stage (between the 8th day until 28th
day of maintenance) is collecting the rabbit production performance data. The data collected are
the measurement of the observed variables: the production/growth performance (feed consumption,
daily gain, feed conversion) and carcass quality (slaughter weight, carcass weight, meat weight,
bone weight, and fat weight). After the data retrieval phase, all rabbits are fausted for 12 hours,
then halally slaughtered by cutting the four channels, (the carotid artery, jugular vein, trachea, and
esophagus). After slaughter, the internal organs was remove off, to obtain the carcass. The data
were analyzed with analysis of variance. The similarity was tested using the Barlett test and the
effect of the treatment was using orthogonal polynomial test.
Results and discussion
Growth Performance
Parameters used in the observation of rabbit growth include feed intake, feed conversion,
and daily gain. Consumption is a basic factor for living and determining production. The results of
the FOS analysis of isolated banana peel (Musa paradisiaca var. Sapientum (L.) Kunt. on New
Zealand White Crossbreed male rabbits in the Table1.
Table 1. The Effect of Treatment on Feed Intake , Daily Gain, and Feed Conversions of New
Zealand White Crossbred Male Rabbit No Variable Treatments
P0 P1 P2 P3 P4
1. Feed Intake
(g/rabbit/day)
84.61** 91.15** 104.76** 108.81** 109.41**
2. Daily gain
(g/rabbit/day)
31.94** 46.30** 52.22** 59.23** 65.20**
3. Feed Conversion 2.67** 1.98** 2.01** 1.84** 1.68**
Note: ** significant difference on level 1%
In the Table 1 shows that the highest average feed intake was achieved by New Zealand White
Crossbred male rabbit receiving P4 treatment (FOS for 28 days), 109.41 g and the lowest was
shown by P0 treatment (without FOS) 84.61 g. The highest daily gain in treatment P4 (65.20
g/rabbit/day) and the lowest on treatment P0 (31.94 g/rabbit/day). While the highest feed
conversion at treatment P0 (2.67) and lowest at treatment of P4 (1.68). The means of ration
consumption 99.75 g was lower than that recommended by Lebas et al. (1996); 110-130 grams for
NZWC rabbits aged 4-11 weeks which were fed balanced diet. Factors affected the ration
consumption level of rabbits, were environmental temperature, health, the feed physical form, food
balance, body weight and growth rate (NRC 1977). The consumption increased, due to the rabbit
trying to meet the energy needs, because the coarse fiber will lowered the energy digestibility
coefficient, thus requiring high energy (Evans, 1981).
406
Lack of crude fiber in rabbit feed can decreased the digestive function, as enteritis (Cheeke
and Patton, 1981), even rabbits can be fed once, twice or three times in a day. Usually rabbit enough
to be fed once a day, and usually should give in the afternoon, because rabbits eat more at night
(Ensminger, 1991). Based on the analysis result, it is known that the treatment has an effect on the
feed intake. A further test using orthogonal polynomial is known that the treatment gives a
quadratic effect on the 1% level, as shown in Figure 1 with the quadratic equation of y = 0.0313x2
+ 1.8358x +83,233. The optimum point was obtained on the 36th day of observation with feed
intake of 110.15 g/rabbit/day. And the determinant correlation (R2) between the influence of the
former feed intake of FOS (98%).
The rate of livestock growth is influenced by the amount, quality of the ration and by the
environmental temperature. The growth pattern will depend on the management system, the
nutritional level of available feeding health and climate (Templeton, 1968). Growth is a change of
elements that includes changes in life weight, shape, linear dimension and body composition. Also
changes the body components such as muscles, fats, bones and organs; and carcasses chemical
components, especially water, fat, protein and ash. The normal growth pattern is a combination of
the growth patterns of all constituent components. Under ideal environmental conditions, the shape
of the post-natal growth curve for all livestock species is similar, following the sigmoid growth
curve pattern. In accordance with the carcass component growth pattern that begins with rapid bone
growth, then after reaching puberty, the rate of muscle growth decreases and the fat deposition
increases.
Fig. 1. Feed intake of rabbit rations of various treatments giving
FOS (Fructooligosaccharide) from banana peel
Sexual puberty is achieved when the reproductive organs have developed and well function
(Blakely and Bade, 1994). During the growth process, livestock is affected by several factors
including genetic, feeding, temperature, adaptability and environment. Sex does not affect carcass
or meat quality but the age (Parigi-Bini et al. (1992 ). Growth is the process of weight gain and
change of body shape and composition, because of the different growth rates of each component.
The growth speed of a young rabbit, are twice the weight of his body every week, so at the age of
three weeks can reach 0.45 kg body weight. After consume solid feed, the growth rate can reach
30 to 50 g/day from the age of three to eight weeks. When breast feeding is the only food consumed,
the growth rate in that period is only 10 to 20 g/day (Spreadbury, 1978), after 10 to 12 weeks of
horizontal growth curve (Lang, 1981); and the results of research obtained is in accordance with,
y = -0.0313x2 + 1.8358x + 83.233
20
40
60
80
100
120
0 7 14 21 28 35fee
d in
take
(g/
rab
bit
/day
)
Treatmeant
407
that the average weight gain 50.98 g/rabbit/day. Further test using orthogonal polynomials, the
treatment gives a quadratic effect at 1% level (Fig. 2).
Fig 2. Daily gain of various treatments giving FOS (Fructooligosaccharide)
from banana peel.
The Fig. 2, shown the quadratic is Y=0.0229x2+1.7754x+32.848, at the optimum point of
the 39th day of observation, the growth of daily gain is 67.26 g/rabbit/day. And the determinant
correlation (R2) between the influence of FOS to the daily gain is 99%. Feed conversion is the
ratio between the amount of feed consumtion to produce one kilogram of slaughter weight (Raharjo
, 2005). Rabbits have a unique behavior that is re-eating feces (coprophagy or caecotrophy), that
rich of vitamin, Niacin, Riboflavin, Pantothenic Acid, Cyanocobalamin (B12), and VFA.
According to Raharjo (2005) the composition of fat, cholesterol and salt are low in rabbit carcasses.
High-quality feeding with good management can result a rabbit feed conversion of 2.80-4.00
(Aritonang et al., 1990). In this study, there was a lower converting mass of 2.04 and a further test
using orthogonal polynomials is known that the treatment gives a quadratic effect at 1% level, as
shown in Figure 3.
Fig. 3. Feed convertion of rabbit rations of various treatment giving
FOS (Fructooligosaccharide) from banana peel
y = -0.0229x2 + 1.7754x + 32.848
10
20
30
40
50
60
70
0 7 14 21 28 35
dai
ly g
ain
(g/
rab
bit
/day
)
Treatment
y = 0.0013x2 - 0.0662x + 2.5858
0.5
1
1.5
2
2.5
3
0 7 14 21 28 35Fee
d C
on
vert
ion
Treatment
408
In Figure 3, the quadratic equation that y = 0.0013x2+0.0662x+2.586, shows the optimum
point of the 26th day of observations 1.74 feed conversion, and determinant correlation (R2)
between the former use of FOS to feed conversion by 94%. In this study, rabbits were given FOS
can increase the feed intake, body weight gain, but has low feed conversion. FOS
(Fructooligosaccharide) is a type of short polysaccharide chain, is fiber food that is undigested that
helps maintain the health of the digestive tract but can be utilized by lactic acid bacteria in the
colon, especially Bifidobacterium sp and Bacteroides sp and will inhibit the growth of pathogenic
bacteria (Yun, 1996 ; Murniasih, 2010), are the source of energy for monogastric animals
herbivores other than carbohydrates, the VFA (Volatile Fatty Acids) such as butyric acid,
propionate and acetate has been absorbed in the digestive tract that will be a source of energy
(Lebas, 1996).
Carcass Production
Slaughter weight (g), carcass weight (g), meat weight (g), bone weight (g), and fat weight
(g) were significantly different at 1% (Table 2). Nevertheless, the values of the four treatments
were increasing in slaughter weight (g), carcass weight (g), carcass percentage (%), and meat
weight (g) but decrease in fat weight (g) treated with FOS and has not difference in bone weight
(g) as shown in Table 2.
Table 2. The Effect of Treatment on Slaughter Weight,
Carcass Weight, Carcass Percentage , Bone Weight, Meat Weight,
and Fat Weight in the New Zealand White Crossbred Male Rabbit.
No Variable Treatments
P0 P1 P2 P3 P4
1 Slaughter Weight (g) 1439.20** 1858.60** 2087.60** 2307.00** 2435.00**
2 Carcass weight (g) 713.2** 946.80** 1086** 1205.60** 1414.40**
3 Carcass percentage (%) 49.6 50.9 52.0 52.3 57.6
4 Bone weight (g) 251.37tn 250.41 tn 251.47 tn 253.33 tn 254.56 tn
5 Meat weight (g) 418.60** 528.00** 762.60** 798.00** 935.00**
6 Fat weight (g) 80.84** 75.48** 73.35** 63.38** 63.24**
Note: tn = non significant, ** = significancy level 1%
The treatment of FOS (Fructooligosaccharide) isolated from banana peel (Musa paradisiaca var.
Sapientum (L.) Kunt, increasing the slaughter weight, carcass weight, carcass percentage, bone
weight, the meat weight, and lower carcass fat weight. Slaughter weight affected by the age, type,
and ration. Young rabbits will produce low slaugher weight compared with the mature rabbit.
Growth can occur due to an increase of the cells number and also the body cell size. The process
occurs in line with the age and condition of the rabbit (Yurmiaty, 2006),. The means of slaughter
weight (2029.08 g), are consistent with Hernandez et al., (2001) which uses four types of rabbit
(California, Chinchilla, New Zealand age 80 days and NZW age 90 days) has the average slaughter
weight (1900-2000 g) and carcass weight (1100 - 1180 g). Based on the result of the analysis, it is
known that the treatment has effect on the slaughter weight. Further trials using orthogonal
polynomials, the treatment is a quadratic on the level 1%, (Figure 4).
409
Fig. 4. Slaughter weight of rabbit rations of various treatments giving
FOS (Fructooligosaccharide) from banana peel
In the Fig. 4, shows the quadratic is y = 0.8111x2+8.082x+1454.4 at the optimum point of
the 35th day, the slaughter weight (2494.20 g). And the determinant corelation of FOS to slaughter
weight is 99% . Carcass weight and carcass percentage is affected by slaughter weight (Metzger et
al., 2003). The carcass weigh, without the blood, head, skin, liver, tail, digestive tract and its
contents, and chest cavity contents, except the kidneys, (Rao, et al. 1978). The highest carcass
weight was obtained in the P4 treatment (1414.40 g), and the lowest in treatment P0 (713.2 g),
while the average carcass weight (1078.4 g). The results is higher than Agustin et al (2017), that
fed of corn oil in the feed (975.51 g). The slaughter weight, higher than the carcass weight. The
carcass production is influenced the slaughter weight. Brahmantiyo et al. (2010) states that the
carcass weight is higher than the slaughter carcass weight. Haryoko and Warsiti (2008), stated that
the carcass component consists of meat, bone and fat. Based on the result of the analysis, it is
known that the treatment has effect on the carcass weight. A further test using orthogonal
polynomial is known that the treatment is linearly at 1% level, as shown in Figure 5, with equation
of y = 20.26x + 797.6. The percentage was influenced by the length use of FOS on carcass weights;
has a determinat correlation (R2) of 95%. Rabbits with heavy body weight can produce a large
percentage of carcasses, the increasing of live weight will increase the feed consumption. Rabbit
consume more feed have a tendency to accumulate more protein as a growth response and increased
the carcass weight. Carcass weight and carcass percentage is highly dependent on breed,
environment, live weight, and nutrient in feed. The percentage of carcass has a positive relationship
to the energy content in the ration.
y = -0.8111x2 + 58.082x + 1454.4
500
1000
1500
2000
2500
3000
0 7 14 21 28 35
Slau
ghte
r w
eigh
t (g
)
Treatment
410
Figure 5. Carcass weight of rabbit rations of various treatment giving FOS
(Fructooligosaccharide) from banana peel
Body size increase proportionally with the body weight of a livestock (Rao et al., 1978).
The carcass percentage is in line with the carcass weight, as the carcass percentage, is the ratio
between carcass weight and weight live when slaughter, multiplied by 100% (Santoso, 2010). In
this study, the highest percentage of carcass was found in the treatment R4 (57.6%), and the lowest
in treatment R0 (49.6%). The carcass percentage, still higher than that of Ozimba and Lukefahr
(1991) which obtained a 55% carcass percentage on NZW, California and California NZW cross
rabbits. The carcass production of New Zealand White rabbit, local rabbit, NZW Crossbred and
Chinchilla crosses, are 45.8, 42.6, 48.9 and 46.7% respectively (Diwyanto et al., 1985).
Commercial carcass production, is strongly influenced by the slaughter weight (Rao et al., 1978),
and the commercial parts of carcass weight, foreleg, rack, loin and hindleg (Blasco et al., 1992).
Conclusion Feeding FOS (Fructooligosaccharide) from the isolation of Ambon banana peel (Musa
paradisiaca var. Sapientum (L.) Kunt., has effected the growth performance (feed intake, slaughter
weight, daily gain, and feed conversion) in quadratic and carcass production (carcass weight, meat
weight, bone weight, and fat weight) linearly in New Zealand White Crossbred male rabbits, and
further studies related to the use of FOS as prebiotics in rabbit feed.
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