Veterinary Research Forum

Abstract Artificial insemination is a well-established and widely used method for genetic improvement in cattle breeding industry. Recently, researchers have shown an increased interest in the cryoprotective effects of minerals and antioxidants on semen. Previous studies on calcium (Ca) and magnesium (Mg), two main macro-minerals, have mainly investigated their roles in mammalian spermatogenesis and fertility. In addition, the experimental data examining the semen content regarding these minerals and antioxidants from different animal species are rather controversial and there is no general agreement about their associations with semen quality. Therefore, this study was conducted to assess the seminal plasma concentrations of Ca, Mg and total antioxidant capacity (TAC) in first and second ejaculations of dual-purpose Fleckvieh bulls and to link them to the sperm characteristics of fresh and frozen-thawed semen. Sperm progressive motility after thawing was used to classify the data into three groups: < 40.00%, 40.00 to 50.00% and > 50.00%. The measurements of two minerals and TAC were carried out using spectrophotometry and enzyme-linked immunosorbent assays, respectively. The results showed that there were significant differences in several parameters of semen quality between first and second ejaculations. No significant differences were also found on Ca and Mg concentrations and Ca/Mg ratio. The TAC level was significantly higher in the first ejaculation than the second one. The findings of this study suggest that TAC is a potential marker for bull semen quality assessment in the frozen semen production industry.

Abstract Canine seminal plasma contains antioxidant enzymes to protect sperm against internally generated ROS. These enzymes are removed from seminal plasma during the process of cryopreservation. The freezing/thawing process can cause some morphological and functional changes via ice crystallization and osmolality imbalance. The present study was conducted to evaluate the effects of curcumin supplementation on sperm total count, motility, progressive motility, viability, morphology, total antioxidant capacity (TAC), DNA integrityand NOX5 gene expression of dog frozen semen. The pooled semen was allocated to fresh (Group 1) and frozen (Group 2) controls, curcumin (2.50 mM) (Group 3) and curcumin (5.00 mM), (Group 4). Sperm parameters including total sperm count, morphology, motility, progressive motility, sperm concentration and DNA integrity in addition to TAC were evaluated in fresh and frozen-thawed semen samples. Real-time PCR was used to investigate NOX5 and GADPH (reference gene) genes expressions. Curcumin at 2.50 mM provided a greater protective effect on the DNA integrity compared to 5.00 mM and control groups. TAC was significantly higher in 2.50 mM group than other groups. NOX5 gene expression in curcumin 2.50 mM was higher than 5.00 mM group. In conclusion, curcumin seems to emolliate sperm parameters and to protect sperm against sperm reactive oxygen stress and increases NOX5 gene expression.

Abstract Testicular torsion is a consequence of spermatic cord twisting which causes progressive damage to the structure of the testis and reduces sperm quality and usually results in infertility. In the present study, with the assumption of the protective effects of L-carnitine and betamethasone against ischemia-reperfusion (IR) injuries, their effects on twisted testicles were evaluated and compared. Twenty Wistar rats were randomly divided into four groups and used in this study. Except for the Sham (S) group, testicular IR was induced surgically in three other groups, including Control (C), Betamethasone (BM), and L-carnitine (LC) groups. Betamethasone and L-carnitine were injected before detorsion in the BM and LC groups, respectively. After twelve hours of reperfusion, the testicles were detached, and prepared for sperm parameters evaluation such as sperm count, motility, viability, morphology, and chromatin quality, and histopathologic evaluations, including mean seminiferous tubular diameter (MSTD), germinal epithelial cell thickness (GECT), and Johnsen’s mean testicular biopsy scoring (MTBS). The MSTD, GECT, and healthy sperms in the C group were significantly lower than the other groups, while the BM and LC groups were significantly different from others in MTBS. The number of sperms and sperm motility in the BM group was significantly higher than the C group. Sperm viability in the BM and LC groups were significantly higher than the C group. The results of this study showed that both L-carnitine and betamethasone similarly can be effective in treating testicular IR injuries.

Abstract The present study aimed to determine the effect of administrating prostaglandin F2α (PGF2α) and GnRH at the time of artificial insemination (AI) on the pregnancy per artificial insemination (P/AI) and the pregnancy survival rate of dairy cows. A number of 830 lactating Holstein cows were randomly divided into four groups. Cows in group 1 (n=200) treated with 150 µg d-cloprostenol. In group 2 (n=212), cows received 10 µg buserelin acetate, and group 3 (n=205) was treated with both 150 µg d-cloprostenol and 10 µg buserelin acetate. In addition, 213 cows were assigned as control group which received normal saline as placebo (group 4). To measure progesterone, milk samples were collected at the insemination day and five days later. Pregnancy diagnosis was performed 28 and 60 days after the insemination, and the size and number of corpus luteum (CL) and twin pregnancies were recorded. Hormone therapies had no effect on the P/AI, pregnancy survival rate, and the size and number of CL. The P/AI ratio in groups 1, 2, 3 and 4 were 38.50%, 42.92%, 41.46% and 40.84%, and the pregnancy survival rates in groups 1, 2, 3 and 4 were 84.42%, 86.81%, 88.23% and 83.91%, respectively. The probability of a twin pregnancy was significantly higher in group 1 (15.58%) than other groups. There was no significant difference between groups in terms of the offspring gender. In conclusion, the administration of d-cloprostenol or buserelin acetate at the time of AI had no effect on P/AI and pregnancy survival rate in dairy cattle under no heat stress condition, while the administration of d-cloprostenol increased the probability of twin pregnancies.

Abstract The aim of the present study was to investigate the diagnostic evaluation of milk lactate dehydrogenase (mLDH) and alkaline phosphatase (mALP) activities by receiver operating characteristic (ROC) analysis curve in early lactation of ewes with subclinical mastitis (SCM) and determine the correlation between number of somatic cell count (SCC) and mLDH and mALP activities. A total of 196 udder half milk samples were collected within the first 6 weeks of lambing. The SCM was determined by positive milk bacterial culture and positive California mastitis test (CMT); SCC was determined by fossomatic method and enzyme activities were determined spectrophotometrically. The mLDH and mALP of SCM cases were positively correlated with SCC values. Values of mLDH, mALP and SCC were significantly higher in SCM than non-SCM udder halves. The optimum cut-off points of mLDH and mALP activities for SCM diagnosis were determined at 203.61 (U L^-1) and 329.84 (U L^-1), respectively. In conclusion, SCC has positive correlation with mALP and mLDH activities in SCM ewes and mLDH and mALP activities could be considered as reliable indicators for intra-mammary inflammation diagnosis.

Abstract Cyclophosphamide is a chemotherapy drug for the treatment of cancer. Chicken embryo amniotic fluid, vitamin C and coenzyme Q10 have anti-oxidant properties. Total of 70 adult female mice were selected and divided into seven groups. The first group that received 2 ml kg^-1 of inactivated amniotic fluid subcutaneously. The second group treated with 75 mg kg^-1of cyclophosphamide by intraperitoneal injection. Third to fifth groups received 1, 2, and 4 ml kg^-1 of chicken embryo amniotic fluid, respectively. The sixth group received vitamin C at a dose of 0.2 mg g^-1 of body weight by oral gavages. Seventh group received 10 mg kg^-1 coenzyme Q10 intraperitoneally. All cyclophosphamide treated groups (3-7) received 75 mg kg^-1 of cyclophosphamide intraperitoneal on day 22. The mice were euthanized on day 29 and ovarian tissue antioxidant enzymes including glutathione peroxidase, superoxide dismutase and catalase activities and malondialdehyde (MDA) were evaluated. Activities of above mentioned enzymes in treatment groups (3-7) was significantly higher than patient control group (2). The results also revealed that MDA levels were higher in the control group in comparison to other treatment groups. Therefore, it is concluded that the chick embryo amniotic fluid and coenzyme Q10 can compete with compounds like vitamin C in increasing the anti-oxidant level in ovarian tissue.

Abstract Busulfan is known to cause several adverse effects including reproductive toxicity in humans. Garlic (Allium sativum), a widely distributed medicinal plant, is highly regarded for its medicinal activities including antioxidant property.This study was conducted to assess whether garlic extract could serve as protective agents against testicular toxicity during busulfan treatment in a mice model.Seventy-two adult male mice were randomly divided into nine groups. In groups 1,2 and 3, distilled water, busulfan, and dimethyl sulfoxide and in the treatment groups hydro-alcoholic extract of garlic was administered orally at different doses per day (groups 4, 5 and 6; 200, 400, 800 mg kg^-1 respectively). Groups 7, 8 and 9 were treated with the extract (200, 400 and 800 mg kg^-1, respectively) plus busulfan. Following euthanasia, blood samples and epididymal sperm were collected.The busulfan-treated group showed significant decreases in sperm qualityparameters, and serum levels of testosterone, LH and FSH was observed in the busulfan-treated mice. In addition, the TAC levels and antioxidant enzymes activities were reduced and malondialdehyde (MDA) levels were increased in the busulfan-treated mice. Notably, garlic extract co-administration caused a considerable recovery in sperm qualityparameters, TAC levels, antioxidant enzymes activities, hormonal changes and MDA level. Based on our results, garlic has antioxidant effects against busulfan-induced testicular damages in mice.

Abstract The aim of this study was to evaluate the effects of ovarian tissue vitrification and two-step in vitro culture on the metaphase II (MII) oocyte reactive oxygen species (ROS) level, mitochondrial transcription factor A (TFAM) expression and succinate dehydrogenase (SDH) activity. After collection of neonatal mouse ovaries, 45 ovaries were vitrified and the others (n = 45) were considered as control. All ovaries were cultured for seven days, and their isolated preantral follicles were cultured in three-dimensional culture system. After 12 days in vitro culture, the follicular development and oocyte maturation were evaluated and compared in vitrified and non-vitrified ovaries. The collected MII oocytes were inseminated with capacitated spermatozoa. The fertilization, embryonic development, ROS level, TFAM gene expression and SDH activity of oocytes were assessed and compared. There was no significant difference between morphology and percentage of normal follicles between vitrified and non-vitrified ovaries at the beginning of culture. The follicular development and hormone level in the vitrified group was significantly lower than non-vitrified group and the ROS concentration in the vitrified group was significantly higher than non-vitrified group after one-week organ culture. After follicular culture, there was no significant difference in follicular development, oocyte maturation, fertilization rate, TFAM gene expression, ROS level and mitochondrial SDH activity between vitrified and non-vitrified groups. This study showed that mouse ovarian tissue vitrification influenced the follicular development through increase in ROS level during organ culture but these harmful effects of vitrification method may be recovered during the follicular culture period. Thus, vitrification and ovarian organ culture method should be improved.

Abstract This study was aimed to investigate the effects of 17 𝛽-estradiol (E2) and 𝛼-zearalenol (α-ZOL) on motility parameters, plasma membrane integrity, levels of produced nitric oxide (NO) and total antioxidant capacity of Ghezel ram sperm during the liquid storage at 4 ˚C, for various periods of time. Semen samples were collected from four rams and diluted with Tris–egg yolk extender and supplemented with E2 (100 µmol) or different concentrations of α-ZOL (100 pmol, 100 nmol and 100 µmol) at a final concentration of 200 × 10⁶ sperm per mL. We failed to show any significant effect of E2 at 100 µmol concentration on ram’s sperm parameters while α-ZOL resulted in a significant decrease of plasma membrane integrity at 100 µmol concentration (55.40% for α-ZOL vs 62.20% for control) after 96 hr incubation. Alpha-ZOL had decreasing effect on sperm motility parameters including curvilinear velocity and average path velocity at 100 µmol concentration after 96 hr storage. Although remarkable reduction of total antioxidant capacity at high concentration of α-ZOL and long incubation time was found, however no significant changes were recorded in NO level during storage time. It was concluded that the detrimental effect of α-ZOL on ram sperm might be attributed to its induced oxidative stress and damage to the plasma membrane.

Abstract This study was carried out to assess the different ovarian transplantation sites after short-time autografting. Female rats were randomized into five groups, with six rats in each group, including control (intact), cervical subcutaneous transplanted (CST), back subcutaneous transplanted (BST), subfascial transplanted (SFT) and intramuscular transplanted (IMT) groups. In all experimental groups, the right ovary was removed and transplanted into different sites. After three weeks, ovaries were removed for morphology assessment, follicular counting and the rates of corpus luteum (CL) and cyst formation. Transplanted ovaries in BST and SFT groups were full of cysts and did not have sufficient numbers of intact follicles and were excluded from experiments. In IMT and CST groups, re-anastomosis, follicular development and good homogeneity of the stromal tissue were seen. However, the difference in intact antral follicles between CST (7.92 ± 0.02%) and CST-Op (opposite ovary of CST group) (30.99 ± 0.03%) was significant as well as the difference between CST (7.92 ± 0.02%) and control (10.08 ± 0.01%) groups. In addition, the number of intact primordial follicles in the CST-Op (16.58 ± 0.02%) group was significantly less than that of the control (40.40 ± 0.03%) group. Interestingly, the number of CL was significantly increased in the CST-Op (11.71 ± 0.01%) and IMT-Op (9.16 ± 0.02%) groups compared to the control and experimental groups. Although both intramuscular and subcutaneous sites effectively preserved ovarian follicles after three weeks, cervical subcutaneous site was better suited for auto-transplantation in rat.

Abstract Busulfan is an alkylating agent affects ovarian follicles growth by oxidative stress induction. Satureja khuzestanica has antioxidant effects. The aim of this study was to examine whether S. khuzestanica essential oil (SKEO) exhibits protective effects on busulfan-induced ovarian failure. Eighty-four adult female mice were divided into six groups including dimethyl sulfoxide (control), SKEO 225.00 mg kg^-1 (orally), busulfan 3.00 mg kg^-1 (orally), busulfan 36.00 mg kg^-1 (intraperitoneally), busulfan 3.00 mg kg^-1 and SKEO and busulfan 36.00 mg kg^-1 and SKEO. After 28 days, the mice were euthanized and oocytes were removed for in vitro fertilization (IVF) rate evaluation. Oocyte quantity and quality, fertilization rate and pre-implantation embryo development were daily examined with a stereo microscope in a period of 120 hr. Serum levels of estradiol and progesterone were also evaluated. Busulfan caused significant decreases in oocyte number and quality, fertilization rate, pre-implantation embryo development and embryo quality. The SKEO significantly decreased the adverse effects of busulfan. The present study indicated that SKEO can protect female fertility potential against busulfan induced damages.

Abstract Bisphenol-S (BPS) is a new bisphenol-A substitute widely used in many plastic products. Bisphenol-A as a main member of bisphenol family has been known as an endocrine system disrupter chemical compound. Like other members of bisphenol family, there is public health concern about the toxic effects of BPS on reproductive system, thus, we examined BPS effects on in vitro fertilization (IVF) potential and oxidative stress status in a murine model. Adult female mice (n = 70) were randomly divided into control and BPS-treated groups. Bisphenol-S was administered at doses of 0, 1, 5, 10, 50 and 100 µg kg^-1 body weight per day intraperitoneally for 21 consecutive days. Twenty-Four hr after the last treatment, five mice in each group were super-ovulated and the oocytes were harvested for IVF. All ovaries were collected and used for biochemical factors analyses. Bisphenol-S exposure at doses more than 10 µg kg^-1 induced developmental arrest of pre-implantation embryos. Further, lipid peroxidation measurement in ovaries indicated that all doses of BPS cause oxidative stress in female mice. In conclusion, BPS administration even in low doses can result in female reproductive toxicities and oxidative stress in mice.

Abstract Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors of transcription factors composed of three family members: PPARα, PPARβ/δ and PPARγ. This study was aimed to evaluate the role of PPARs in the estradiol production via follicle stimulating hormone (FSH) in the ovine Sertoli cells. At the first step, transcripts of PPARα, PPARβ /δ and PPARγ were evaluated by quantitative real time PCR (qRT-PCR) in the ovine Sertoli cells in vitro after FSH treatment. PPARγ transcript was increased in FSH-treated cells while PPARα and PPAR β /δ transcripts were unchanged. At the second step, Pioglitazone as PPARγ agonist and 2-chloro-5-nitrobenzanilide (GW9662) as PPARγ antagonist were used in the FSH-treated Sertoli cells and then, the estradiol production and aromatase transcript were evaluated. Aromatase transcript was increased by pioglitazone in the FSH-treated Sertoli cells while GW9662 did not change its transcript. The estradiol production was increased by low concentrations of pioglitazone in FSH-treated Sertoli cells while the production of this hormone was decreased by the high concentration of Pioglitazone. The GW9662 did not change the production of estradiol in FSH-treated Sertoli cells. It is concluded that FSH regulates the estradiol production and aromatase expression in a way independently of PPARβ/δ and PPARα activation, although FSH increases the transcript of PPARγ and in this way, it could affect (mostly increase) aromatase transcript and estradiol production. Probably, this effect of FSH in the estradiol production via PPARγ is only a servo-assist mechanism which if it was inhibited, the estradiol production was not considerably affected.

Abstract There is no prospective study from Iran to estimate the direct risk of Neospora caninum for pregnancy loss or reproductive factors. In addition, there is no report in the literature concerning the association of N. caninum with dystocia and sex of calves. Therefore, this study was conducted on a group of dairy cows in a large intensive production system during 2011 to 2013 in southern Iran to evaluate the impact of neosporosis on reproductive performance. A total of 253 cows which were diagnosed as pregnant during the first six months of the study were followed until calving or abortion. Reproductive data were collected and N. caninum serostatus was determined using ELISA. To investigate the association of abortion with N. caninum,survival analysis was performed using Cox proportional hazard model. The N. caninum seroprevalence in the study group was 30.40% (95% CI: 27.40, 36.10). The overall abortion rate was 12.25%, significantly higher in seropositive animals (20.80%) than seronegative ones (8.50%). Results of Cox model showed that serostatus of animal for N. caninum and season had significant associations with abortion (p < 0.01). Neospora caninum did not show significant association with other factors such as dystocia and sex of calves. In conclusion, neosporosis is responsible for 12.00% excess abortion risk in infected group and more than 30.00% of abortions could be preventable by control of Neospora in study population. Therefore, control of N. caninum would reduce the economic losses caused by parasite mainly due to pregnancy loss.

Abstract The objective of this study was to highlight the postnatal development of the uterine tubes of the West African Dwarf goat from birth to 28 weeks of age by gross examination and light microscopy. There was a caudal migration of the paired uterine tubes from behind the paired kidneys at birth to the pelvic inlet at week 8 of age. Each uterine tube exhibited three segments namely; infundibulum, ampulla and isthmus. A marked flexure, the utero-tubal junction, was the point at which the uterine tubes joined the uterine horns. The length and absolute weight of the uterine tubes increased from 4.95 ± 0.28 cm and 0.02 ± 0.01 g at birth to 14.98 ± 2.79 cm and 0.22 ± 0.03 g at week 28 of age, respectively. The mucosa of the infundibulum and the ampulla showed long, branched and anastomosing primary, secondary and tertiary mucosal folds which decreased in height towards the isthmus. The mucosal folds within the isthmus were short and lacked the anastomosing pattern. The epithelia of all three segments were pseudostratified columnar. Numerous secretory blebs and extruded nuclei became apparent from week 16 of age. The thickness of the tunica muscularis varied with the segments.

Abstract Testosterone is believed to play a significant role in spermatogenesis, but its contribution to the process of spermatogenesis is not completely understood. Given that extracellular matrix (ECM) facilitates differentiation of spermatogonial stem cells (SSCs) during culture, the present study was conducted to elucidate whether testosterone contribute to the permissive effect of ECM on SSCs differentiation. In experiment 1, testosterone production was measured in testicular cells cultured for 12 days on ECM or plastic (control). In experiment 2, testosterone production was assessed in testicular cells cultured on ECM or plastic (control) and exposed to different concentrations of hCG. In experiment 3, the gene expression of factors involved in testosterone production was analyzed. Testosterone concentration was lower in ECM than in the control group in experiment 1 (p < 0.05). In experiment 2, testosterone concentration was increased in response to hCG in both groups but cells cultured on ECM were more responsive to hCG than those cultured on plastic (p < 0.05). In the experiment 3, qRT-PCR revealed the inhibitory effect of ECM on the gene expression of steroidogenic acute regulatory protein (StAR) (p < 0.05). Nevertheless, the expression of LH receptor was greater in ECM-exposed than in unexposed cells (p < 0.05). In conclusion, the present study showed that inhibiting the expression of StAR, ECM could lower testosterone production by Leydig cells during in vitro culture. In addition, the results indicated that ECM could augment the responsiveness of Leydig cells to hCG through stimulating the expression of LH receptor.

Abstract The transcriptional factor OCT4 regulates pluripotency of stem cells and has an important role during oocyte growth. Whereas, its role has remained ambiguous in ovarian tissue during reproductive cycle. Therefore, this study was aimed to investigate the expression patterns of OCT4 in mouse ovaries during the normal estrous cycle. Adult National Medical Research Institute mice were classified as proestrous, estrous, metestrous and diestrous on the basis of vaginal smear cytology. Their ovaries were removed and the protein and gene expression levels of OCT4 were assessed using immunohistochemical staining and real-time quantitative reverse-transcription PCR, respectively. Immunohistochemical staining revealed the expression of OCT4 in the cytoplasm of corpus luteum cells. In the follicles, OCT4 was expressed in the cytoplasm of granulosa cells. Furthermore, the gene expression levels of OCT4 was significantly higher in the proestrous phase than in the other phases of the estrous cycle (p < 0.05). The results indicated that OCT4 gene expression levels are affected by the cyclic pattern of the estrous cycle.

Abstract Enrichment of cell suspension with germ cells prior to injection into recipient seminiferous tubules is of importance in spermatogonial stem cells (SSCs) transplantation. Knock-out serum replacement (KSR) has been reported to enhance the proliferation of murine SSCs and human embryonic stem cells. The aim of the present study was to investigate the effect of KSR versus fetal bovine serum (FBS) and their interaction on colonization of bovine SSCs in vitro. When FBS (10%) was replaced with KSR (10%), a significant increase in the colonization of SSCs and the expression of Thy1, as marker for enrichment of SSCs, was observed. It was revealed that the lesser proliferative effect of FBS as well as the greater proliferative impact of KSR on SSCs colonization were not irreversible as cells having been cultured with FBS (10%) for three days with low colonization showed high rate of colonization in response to KSR (10%) and cells having been cultured with KSR (10%) with high colonization experienced low rate of colonization in response to FBS (10%). Further, it was shown that FBS did not contain factors inhibiting SSCs colonization and it simply lacked factors essential for SSCs proliferation because the combination of FBS (5%) and KSR (5%) resulted in even greater rate of colonization than did KSR (10%). In conclusion, the present study showed that addition of KSR to culture medium would significantly increase SSCs proliferation.

Abstract In the present study, the effect of intrauterine infusion of Zataria multiflora extract on the clinical endometritis was investigated. Vaginal examination, transrectal palpation and ultrasonography were used to inspect the genital tract at 30-40 days in milk and two weeks later the same approach was applied. Cows with clinical endometritis were randomly divided into three treatment groups: Z. multiflora extract (n = 56), penicillin + streptomycin (pen + strep, n = 55), and placebo (n = 20). Cervical cytology, reagent strip test and cell counting by means of Neubauer hemocytometer were carried out in both examinations. Clinical cure rate of cows with endometritis of score 1 were 45.5, 34.5 and 53.6% in placebo, pen + strep and Z. multiflora, respectively. Clinical cure rate of cows with endometritis of score 2, 3 were 66.7, 84.6 and 56.0% in placebo, pen + strep and Z. multiflora, respectively. Overall, proportions of successfully treated cows were 55.0, 58.2 and 54.7% in placebo, pen + strep and Z. multiflora, respectively (p > 0.05). In placebo, none of the parameters were significantly different between first and second examination, while we found the significant differences in percentage of neutrophils and leukocyte esterase activity in other groups (p < 0.05). First service conception rate of cows was higher in Z. multiflora compared to other groups; however, this difference was not significant. In conclusion, pen + strep and Z. multiflora extract can be effective on the clinical endometritis and may improve reproductive performance. The extract of Z. multiflora can be useful as an alternative therapy for treatment of clinical endometritis in lactating dairy cows.

Abstract It was assumed that uterine stem cells are responsible for the unique regenerative capacity of uterine. Therefore, the aim of the present study was to investigate the expression of the pluripotent stem cell markers in the mice uterine tissue during different stages of estrous cycles. Twelve virgin female NMRI mice (6 to 8 weeks old) were considered at proestrus, estrus, metestrus and diestrus according to the cell types observed in the vaginal smear and underwent hysterectomy operation. Quantitative real-time polymerase chain reaction (PCR) and immunohistochemical staining for pluripotent stem cell markers (SOX2, OCT4, KLF4, and NANOG) were performed. Immunofluorescence staining revealed that expression and localization of the pluripotency markers SOX2, OCT4, KLF4, and NANOG at the protein level were not different throughout estrous cycle. Also, mRNA of pluripotency markers was detected in all tested samples. However, there were no significant differences in their genes expression at each stage and during the estrous cycle. Different hormonal profile during the estrous cycle could not affect expression of pluripotent stem cell markers in uterine tissue.

Abstract Cryopreservation has the capacity to extend spermatozoa’s lifespan and viability. In addition, the semen samples can be collected, preserved and stored or sent to distant locations and still be used long after the death of the semen donor. In this study for the vitrification of dog sperm (fresh and swum-up sperm), different cryopreservation mediums on the basis of glycerol, milk and egg yolk were used. Then, all of the samples were vitrified in the liquid nitrogen and thawed at least 48 hr later for re-examination of sperm parameters. The sperm parameters before and after cryopreservation in all groups were compared. It was found that during vitrification process, spermatozoa were damaged by the mechanical blows in centrifugation during swim-up processing, so they had less resistance than fresh semen. The examination of different cryoprotectants revealed that milk has better effects on the cryopreservation of semen than glycerol and egg yolk. With the comparison of the effects of glycerol and egg yolk as cryoprotectants, it was found that glycerol had better effects than egg yolk on the cryopreservation of the semen. In conclusion, milk might be used as a cryoprotectant instead of glycerol for canine sperm cryopreservation.

Abstract Great apes are mammals close to humans in their genetic, behavioral, social and evolutionary characteristics and new genomic information is revolutionizing our understanding of evolution in primates. However, all these species are endangered. While there are many global programs to protect these species, the International Union for Conservation of Nature (IUCN) projects that in a near future the wild populations will decrease significantly. Nowadays, the relevance of captive populations of great apes is becoming critical for research and understanding of pathophysiology of diseases. In this report, the evaluation of infertility in a group of captive chimpanzees maintained at Leon’s Zoological Park using a human infertility protocol is described. Our results suggested that infertility in this group was due to low hormonal levels and sperm alterations in the male characterized by hormonal assessment and a sperm sample obtained by electroejaculation and cryopreserved using human protocols. In the females, it was demonstrated that it is possible to follow the follicular cycle using non-invasive methods based on morphological changes in genitalia, detection of blood in urine and measurement of hormones in saliva samples; concluding that fertility in females was normal. Also, we demonstrate that human artificial insemination procedures may be applied. Our human approach was successful in finding the infertility cause in this group of captive chimpanzees. In countries with limited resources, collaboration of zoos with human infertility clinics can be beneficial for research and management of reproductive aspects of great apes.

Abstract Taraxacum officinale has been used in Jordan folk medicine to treat male infertility. A recent study has proved a contradictory effect of the whole plant aqueous extract. The aim of the current study was to determine if the leaves of T. officinale have similar anti-fertility activities, and whether this effect is mediated through the regulation of spermatogonial stem cells (SSCs). Fifty adult male rats were divided into five groups. Two groups were gavaged with 1/10 of LD₅₀ of T. officinale whole plant (1.06 g kg^-1 body weight) or leaves (2.30 g kg^-1 body weight) aqueous extract; while two groups were gavaged with 1/20 of LD₅₀ of T. officinale whole plant (2.13 g kg^-1) or leaves (4.60 g kg^-1) extract. The control group received distilled water. Oral administration of T. officinale (whole plant and leaves aqueous extract) caused a significant decrease in testis and seminal vesicle weight, a reduction in serum testosterone concentration, impaired sperm parameters, and a decrease in pregnancy parameters. Testicular histology of treated rats showed structural changes such as hypoplasia of germ cells, reduction in the thickness of germinal epithelium, arrest of spermatogenesis at spermatid stage (late maturation arrest) and reduction in the number of Leydig cells. Gene expression levels of two SSCs markers (GFRα1 and CSF1) responsible for self-renewal were relatively counter-balanced. In conclusion, T. officinale whole plant and leaves aqueous extracts changed the gene expression of two SSCs markers leading to the imbalance between spermatogonia self-renewal and differentiation causing late maturation arrest.

Abstract Leptin, the 16-kDa product of the obese (ob) gene, primarily secreted from adipose tissue, has been implicated to play an important role in the regulation of food intake and energy expenditure. This study investigated protective effect of leptin on trichostatin A-induced apoptotic on in vitro maturation ratio of buffalo oocytes. Ovaries were collected from abattoir and were transported immediately to the laboratory by a thermos flask containing sterile normal saline with antibiotics. Oocytes were aspirated from 2 to 8 mm visible follicles. Oocytes were placed in a culture plate and then incubated at 38.5 ˚C with 5% CO₂ in air for 24 hr. The maturation of oocytes was evaluated under a stereomicroscope. The FITC-Annexin V and propidium iodide staining method was used to detect oocyte apoptosis. In leptin treated groups with 0, 10, 50 and 100 ng mL^-1 and groups that apoptosis was induced, the percentage of oocytes maturation was 77.03, 86.12, 85.08, and 79.89% and 59.96, 56.93 and 51.98, respectively, while the percentage of apoptosis was 8.83, 7.90, 8.58, and 9.39%, and 10.37, 11.57 and 12.03, respectively. Our findings showed that addition of 10 and 50 ng mL^-1 leptin to IVM medium of buffalo oocytes could increase oocyte nuclear maturation, and could decrease oocyte apoptosis when trichostatin A added for inducing apoptosis.

Abstract The present study was conducted to evaluate the effect of fish oil supplementation prior to mating on secondary sex ratio of pups (the proportion of males at birth) in bitches. Sixty five bitches (German Shepherd, n = 35; Husky, n = 30) were enrolled in the study. Bitches (140-150 days post-estrus) were given 2% per dry matter intake palm oil and fish oil in the control (n = 33) and treatment (n = 32) groups, respectively. To induce estrus, bitches were received equine chorionic gonadotropin (eCG) administration (50 IU kg^-1) 30 days after nutritional supplementation followed by human chorionic gonadotropin (hCG) administration (500 IU per dog) seven days later. Bitches were introduced to dogs of the same breed after hCG administration. The weight of bitches was increased over time (p < 0.05), but their weight change was not different between two groups (p > 0.05). The mating rate, pregnancy rate and litter size were not influenced by treatment and breed. Secondary sex ratio was higher in the treatment (105/164; 64.00%) than in the control (68/147; 46.30%) group (p < 0.05; adjusted odds ratio = 2.068). Moreover, secondary sex ratio was higher in Husky bitches (88/141; 62.40%) compared to German Shepherd (85/170; 50.00%; p < 0.05; adjusted odds ratio = 1.661). In conclusion, the present study showed that inclusion of fish oil in the diet of bitches prior to mating could increase the proportion of male pups at birth. In addition, it appears that there might be variation among dog breeds with regard to the sex ratio of offspring.