Veterinary Research Forum

Abstract This study evaluated the protective effects of intraperitoneal tannic acid (TA) against ischemia-reperfusion (I/R) injury in a rat model of testicular torsion. Eighteen adult male Wistar rats were randomized into three groups (n=6 each): sham (surgery without ischemia), I/R (3 hr ischemia + 3 hr reperfusion), and I/R+TA (TA 50 mg kg-1; 100 µL i.p. 30 min before reperfusion). Testicular tissues were sampled immediately after reperfusion for biochemical assays to measure malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GPx) levels. Epididymides were analyzed 60 days later for sperm count, motility, viability, morphology, and DNA integrity. I/R significantly elevated MDA and sperm DNA damage while reducing SOD, GPx, sperm parameters, testicular weight and spermatogenesis. TA administration reversed these changes and restored the parameters to levels close to those of the sham group. Overall, intraperitoneal TA mitigated I/R-induced oxidative stress and preserved reproductive function, indicating its potential therapeutic value in testicular torsion.

Abstract Abstract
The excessive production of reactive oxygen species during the freezing and thawing process triggers lipid peroxidation in the sperm membrane, resulting in oxidative harm and a decline in semen quality. The aim of this study was to examine the impact of different concentrations of canthaxanthin on the quality of goat spermatozoa throughout the process of cryopreservation. Semen was collected from each goat using an artificial vagina for a continuous duration of three weeks. The semen was divided into five equal portions and mixed with an extender having different concentrations of canthaxanthin: 0.00 µM (control), 5.00 µM, 15.00 µM, 25.00 µM, and 35.00 µM. The computer-assisted semen analyzer was used to determine the sperm motility and kinetic parameters. The findings indicated that the inclusion of canthaxanthin at a concentration of 5.00 µM resulted in a minimal enhancement in multiple factors associated with sperm motility, such as overall motility, progressive motility, fast progressive motility, curve-line velocity, distance curve line, amplitude of lateral head displacement, beat-cross frequency, wobble, linearity, and straightness. Nevertheless, these enhancements did not demonstrate statistical significance compared to the control, 15.00 µM, 25.00 µM, and 35.00 µM. The introduction of 5.00 µM of canthaxanthin resulted in a significant rise in straight-line velocity, average path velocity, and head activity compared to the control group. Hence, the addition of 5.00 µM of canthaxanthin could be employed to improve the cryopreservation of goat spermatozoa.

Abstract Bovine viral diarrhea virus is a pestivirus of the Flaviviridae family including two biotypes, cytopathic (CP) and non-CP (NCP). This study aimed to evaluate DNA damage and apoptosis in Madin-Darby bovine kidney cells following infection with both biotypes. The MDBK monolayers were inoculated with a final dose of virus (1.00 × 10³ Tissue Culture Infectious Dose 50% mL-1) and incubated for 24 hr. DNA strand integrity was assessed using alkaline single-cell gel electrophoresis, and DNA damage was quantified through tail moment and olive tail moment indices (n = 3). Apoptosis was evaluated using annexin V–fluorescein isothiocyanate/propidium iodide flow cytometry to determine early and late apoptotic cell populations. Both biotypes significantly increased DNA fragmentation compared to the control group. The tail moment values were 15.89 ± 2.13 (control), 57.63 ± 16.20 (NCP), and 68.15 ± 9.93 (CP); while, olive tail moment values were 8.71 ± 1.01 (control), 29.35 ± 9.18 (NCP), and 35.14 ± 6.90 (CP). Apoptosis analysis showed a higher percentage of apoptotic cells in infected groups, with CP biotype of bovine viral diarrhea virus inducing the greatest early and late apoptotic responses, being consistent with its CP nature. Overall, both biotypes caused notable genomic injury and apoptosis in Madin-Darby bovine kidney cells, with CP producing the highest level of damage, confirming single-cell gel electrophoresis combined with apoptosis assays as sensitive tools for detecting virus-mediated genomic instability and supporting their potential application in breeding programs aimed at enhancing resistance to infectious diseases.

Abstract This study aimed to explore whether melatonin protects TM3 Leydig cells from cobalt (II) chloride (CoCl₂)-induced hypoxia through the transforming growth factor beta (TGF-β) signaling pathway. Cells were divided into four groups: a control group without treatment (Group 1), a melatonin group (10.00 ng mL-1; Group 2), a group treated with CoCl2 (100 µM) to induce hypoxia (Group 3), and a melatonin + CoCl₂ group (Group 4). After 96 hr of incubation, cell viability was assessed using the MTT assay, and transforming growth factor beta 1, activin receptor-like kinase-5, and bone morphogenetic protein 4 gene and protein expressions were measured through RT‑PCR and western blotting. The CoCl₂ and melatonin + CoCl₂ groups exhibited significantly diminished cell viability compared to the control. However, melatonin treatment enhanced survival in the CoCl₂-exposed cells. Notably, transforming growth factor beta 1 expression was elevated in all groups. Activin receptor-like kinase-5 (gene and protein expression increased in CoCl₂-treated groups but was lower in the melatonin + CoCl₂ group. Melatonin treatment reduced bone morphogenetic protein 4 expression compared to the control, while CoCl₂ groups showed increased bone morphogenetic protein 4 levels. These findings suggest melatonin's potential as a therapeutic agent against oxidative stress and hypoxia in TM3 cells through its antioxidant properties.

Abstract Cadmium (Cd) is a highly toxic environmental pollutant known to cause severe damage to the male reproductive system. This study aimed to investigate the protective effects of bee bread (BB), a natural product with anti-oxidant, anti-apoptotic, and anti-inflammatory properties, against Cd-induced testicular toxicity in male Wistar rats. A total number of 32 rats were divided into four groups, including control, BB (0.50 g kg^-1), Cd (5.00 mg kg^-1), and Cd + BB (5.00 mg kg^-1 and 0.50 g kg^-1, respectively) groups. Administrations via oral gavage were performed for 4 weeks. Semen analysis revealed significant reductions in sperm motility and density along with increases in abnormal and dead sperm ratios in the Cd and Cd + BB groups compared to controls. Histopathological examination showed severe degeneration and desquamation of germ cells, tubular atrophy, and a decrease in spermatozoa in the Cd-treated groups. Polymerase chain reaction analysis indicated up-regulation of apoptotic markers (caspase-3, -8, and -9) and oxidative stress enzymes (catalase and superoxide dismutase) in the Cd group, signifying disrupted testicular function. The BB administration partially mitigated Cd-induced damage as evidenced by less severe histopathological changes and moderated gene expression alterations. However, the protective effects of BB were not sufficient to completely counteract the toxic impact of Cd. The present study concluded that while BB had potential in reducing Cd-induced testicular toxicity, its protective efficacy was limited, warranting further research to explore its therapeutic potential in combination with other protective agents.

Abstract The effects of substituting whole egg yolk (WEY) in a tris-citrate-based extender with two derived fractions, including a buffer-soluble fraction (BSF) and an ammonium sulfate insoluble yolk fraction, on the freezability of bull sperm were investigated. The BSF and ammonium sulfate insoluble yolk fraction levels of egg yolk in the respective diluents were consistent with their extracted values from an equivalent volume (20.00%) of egg yolk in the control diluent. The extenders were then enriched with 0.00, 5.00, and 10.00% (v/v) of pasteurized skim milk. Semen samples were collected weekly over five consecutive weeks from six adult bulls, and the standard ejaculates were pooled. The pooled semen was subsequently divided into seven experimental extenders and frozen in 0.50 mL French straws. Various sperm quality parameters, including kinematics, acrosome integrity, capacitation, and DNA fragmentation, were evaluated post-thawing. Results indicated that sperm kinematics and the percentage of acrosome-reacted sperm in experimental extenders were not significantly different from those in the control group. The milk-free ammonium sulfate insoluble yolk fraction extender exhibited higher percentages of capacitated sperms, but lower percentages of spermatozoa with intact DNA compared to the WEY extender sperm. The addition of 10.00% milk into the BSF diluent resulted in a significant increase in the proportion of sperm with intact DNA and a notable decrease in the percentage of sperm with partially fragmented DNA compared to the control. In conclusion, a BSF extender enriched with 10.00% cow's skimmed milk is recommended as a substitute for WEY in the cryopreservation of bull semen. 

Abstract The aim of this study was to investigate the effects of post-partum (PP) periods of different lengths on super-stimulatory and super-ovulatory responses, as well as the number and quality of embryos in Simmental cows. Fifty cows were divided into five groups based on their PP days, including 50 - 60 days (n = 5), 61 - 90 days (n = 17), 91 - 120 days (n = 9), 121 - 150 days (n = 9), and 151 - 420 days (n = 10). On a random day of the estrous cycle, all follicles larger than 5.00 mm on the ovaries were aspirated and a progesterone-releasing device was inserted into the vagina of all donors. Approximately 36 hr after follicle aspiration, all cows were administered 500 µg of follicle-stimulating hormone in decreasing doses, twice daily for 6 days. Ovaries were evaluated using trans-rectal ultrasonography during human chorionic gonadotropin treatment and after embryo collection to assess follicles and corpora lutea, respectively. Super-stimulatory and super-ovulatory responses, as well as embryo yield and quality were found to be similar among cows in the PP days groups of 61 - 90, 91 - 120, 121 - 150, and 151 - 420. However, the embryo recovery rate and mean number of transferable and freezable embryos were adversely affected in cows at 50 - 60 days PP. The findings of our study suggested that day 60 PP served as a threshold for in vivo embryo production in Simmental cows.

Abstract Reproductive failure is the major problem in modern pig production, significantly affecting economic productivity. This study investigated factors associated with reproductive failure in the hyper prolific pig farms, focusing on abortion, mummification, stillbirth and sudden increased sow death. Seven individual sows with reproductive failure clinical signs were surveyed to evaluate the infection rate with related pathogens. Results showed the highest frequency of Mycoplasma suis infections with reproductive problems along with blood samples being 100% positive, fetal mummification rate of 33.33% and stillbirth rate of 25.00%. Additionally, 28.57% of the total blood samples exhibited porcine circovirus type 3 infection, while other pathogens were not detected. The percentage of concurrent infections with M. suis and porcine circovirus type 3 in reproductive sow was 28.57%. The partial 16S rRNA Porcine Haemoplasma is a trivial name for haemotropic Mycoplasma spp., which can attach to the surface of red blood cells leading to deformity and anaemia in a wide range of mammalian animals including pigs. Haemoplasma sequences in this study was absolutely clustered into Haemominutum group. This study demonstrated the first case of acute reproductive disorder due to M. suis reported in imported hyper prolific sows.

Abstract Anti-oxidants are vital for protecting sperm and can mitigate the negative effects associated with cryopreservation. This study was conducted to evaluate the protective effects of adding anti-oxidants (vitamins and amino acids) to extender in sex-reversed rainbow trout (Oncorhynchus mykiss) sperm. The collected sperm was diluted at the ratio of 1 : 5 by the extenders supplemented with different anti-oxidants including 1.00 mM of ascorbic acid  and L-tryptophan, 2.00 mM of cysteine and α-tocopherol, and their combination. After dilution, the semen was aspirated into 0.50 mL straws, and the straws were placed on the tray, frozen for 10 min, and plunged into liquid nitrogen. Straws were thawed in a 30.00 ˚C water bath for 15 sec. The Sperm Class Analyzer System was used to evaluate sperm kinematics. The activity of anti-oxidant enzymes and lipid peroxidation were determined as oxidative stress indices. Our data indicated that the incorporation of anti-oxidants and amino acids increased sperm motility duration. The elevated activity of glutathione peroxidase, superoxide dismutase, and catalase in post-thaw samples indicates that the anti-oxidant system in sex-reversed rainbow trout sperm likely plays a crucial role in protecting membrane compounds from oxidation. In conclusion, the combination of 1.00 mM L-tryptophan and ascorbic acid to the extender media caused a prolonging effect in sperm motility after thawing and they have the potential to serve as effective agents for improving sperm cryosurvival.

Abstract Molar pregnancy is a rare condition in women, referring to the development of fetal membranes with or without an embryo. A 22-year-old mare referred to the veterinary faculty clinic due to the delayed parturition. In the ultrasound examination, the uterine membranes were seen suspended in fluids without any trace of the fetal organs. The mare underwent caesarean section, a mixture of brown fluid with fragments of fetal membranes was removed, but no fetus or any remnants of the fetus were found in the uterus. In the histopathological examinations, the fetal membranes showed severe degenerative changes. Also, the thickness of the endometrium had lost its normal structure and the layers of stratum spongiosum and stratum compactum could not be separated. The contents of the uterus were evacuated as much as possible and the mare regained her full health after the surgery. This is the first case of continued pregnancy without an embryo in a horse, which can be an unusual type of molar pregnancy.

Abstract This study compared the effects of a dose of human chorionic gonadotropin (hCG) administration at the time of insemination on the reproductive outcome of estrus-synchronized Mongolian ewes subjected to two artificial insemination methods during the breeding season. All females were treated with a polyurethane intravaginal sponge impregnated with 45.00 mg of flurogestone acetate for 12 days, followed by an intramuscular injection of 330 IU of equine chorionic gonadotropin at sponge removal. In Experiment 1, 150 ewes were inseminated using a laparoscopic intrauterine artificial insemination method 48 hr after sponge removal. The ewes were randomly assigned to the hCG group which received 500 IU of hCG at the time of insemination, and the control group which received 1.00 mL of sterile saline solution. In Experiment 2, 85 ewes were inseminated twice at 48 and 60 hr after sponge removal using a cervical artificial insemination method. The ewes were randomly assigned to the hCG group, which received 500IU of hCG at the time of the first insemination and the control group which received 1.00 mL of sterile saline solution. The pregnancy rate in the control group was not significantly different compared to the hCG group in Experiment 1 and the pregnancy rate in the control group was significantly higher compared to the hCG group in Experiment 2. In conclusion, the administration of hCG at the time of insemination could not be recommended in ewes when implementing a fixed time artificial insemination protocol during the breeding season.

Abstract This study examined the effects of alpha-ketoglutaric acid (AKG; 2.00, 4.00, and 8.00 mM L^-1) and folic acid (FA; 50.00 nM L^-1) on bull sperm cells under ammonia stress induced by 175 µM L^-1 ammonium chloride. Sperm parameters including kinematic motility, survival rate, membrane integrity, DNA integrity, superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), total antioxidant capacity (TAC) activities, and apoptosis were assessed in the groups. Sperm motility indices, survival rate, plasma membrane integrity, SOD, CAT, TAC, and GPx enzymes activity, DNA damage, and apoptosis in the treated groups were significantly differed from those in the control groups. The AKG concentrations of 4.00 and 8.00 mM L^-1 co-supplemented with 50.00 nM L^-1 FA mitigated the negative effects of ammonia on sperm cells. This study indicated that supplementation with AKG and FA at the desired concentrations counteracted the adverse effects of ammonia toxicity that preceded clinical signs. Further studies are needed to evaluate the fertility of these sperms, either in vitro or in vivo.

Abstract Leydig cells play a crucial role in male reproductive physiology, and their dysfunction is often associated with male infertility. Hypoxia negatively affects the structure and function of Leydig cells. This study aimed to investigate the impact of melatonin on the c-Jun N-terminal kinase (Jnk), P38, and extra-cellular signal-regulated kinases 1 and 2 (Erk1/2) mitogen-activated protein kinase (MAPK) signaling pathways in TM3 mouse Leydig cells under hypoxia induced by cobalt (II) chloride (CoCl₂). The TM3 cell line was utilized as a subject of research, and 100 μM CoCl₂ was employed to induce hypoxia. Following the addition of 10.00 ng mL^-1 melatonin, quantitative reverse transcription-polymerase chain reaction and western blot analyses were conducted to assess the gene expression and protein level of Jnk, p38, and Erk1/2, while enzyme-linked immunosorbent assay was used to measure testosterone secretion. The results showed that melatonin significantly increased testosterone production in the CoCl₂ + melatonin group compared to the CoCl₂-treated group. Furthermore, melatonin elevated both the protein level and mRNA expression of Erk1/2, Jnk, and p38 genes in the CoCl₂ + melatonin group compared to the CoCl₂ group. In conclusion, melatonin activated the Jnk, p38, and Erk1/2 MAPK signaling pathways and enhanced testosterone production in the presence of CoCl₂ in TM3 cells.

Abstract The cooling procedure markedly diminishes the quality of guinea pig (Cavia porcellus) sperms, primarily because their membranes are highly susceptible to this process. This susceptibility triggers the generation of reactive oxygen species and free radicals, ultimately leading to lipid peroxidation in the sperm membrane. Surprisingly, there has been a lack of research on the use of Tris-based extenders to safeguard guinea pig sperm under refrigeration conditions. This study aimed to assess the viability of guinea pig spermatozoa diluted in Tris buffer-based extenders during storage at 5.00 ˚C. Sperm collection was carried out through castration of the animals. For this study, 10 adult male guinea pigs were utilized, being divided into four groups including phosphate-buffered saline (PBS), human tubal fluid (HTF), Tris-citric-glucose (TCG), and Tris-fructose-yolk (TFY) cultures. Evaluations including sperm motility, morphology, plasma membrane integrity, viability, and total count were conducted at 0, 24, and 48 hr after sampling. The results obtained indicated that at the 24-hr and 48-hr marks of the experiment, both overall and progressive motility percentages, viability, plasma membrane integrity, and morphology of sperms in the PBS and HTF cultures exhibited a significant increase in comparison with the TCG and TFY cultures. Consequently, it can be inferred that PBS and HTF cultures are highly effective in preserving the quality of guinea pig spermatozoa.

Abstract During the freezing process of semen, due to the generating of significant amounts of free radicals, the quality of sperm changes. Epigallocatechin-3-gallate (EGCG) is a green tea catechin, which in this study was applied to investigate its effect on the quality of bulls' sperm. We collected semen samples with an artificial vagina from 12 Simmental bulls to evaluate the effect of EGCG (10.00 and 20.00 µmol) in two cryopreserving methods on the quality parameters of semen. We designed six groups including two control groups (method one and two) and four treatments (EGCG 10.00 µmol + method one; EGCG 20.00 µmol + method one; EGCG 10.00 µmol + method two; EGCG 20.00 µmol + method two). The 20.00 µmol EGCG and a method two significantly affected the amending oxidative conditions as well as an increase in total antioxidant capacity and a decrease in malondialdehyde. The effect of EGCG in both concentrations was more on method two. The desired impact on sperm motility, viability, inhibition of lipid peroxidation and sperm DNA damage was observed in EGCG groups compared to control groups. Among the two methods, the method two had fewer adverse effects on the plasma membrane, motility parameters, viability and DNA of sperm. The EGCG in the semen extender yielded a favorable impact on thawed sperm. This effect was prompted in combination with the method two.

Abstract The Cholistani cow is considered one of the milch breeds of Zebu cattle. Despite being heat and tick-resistant, it has not yet gained much popularity among Pakistan dairy farmers due to its long calving interval. This study aimed to understand the basic reproductive physiology of Cholistani cows using sonography and reproductive biotechnologies such as estrus synchronization and timed artificial insemination to improve reproductive efficiency. In experiment 1, six Cholistani cows with mixed parity 3.20 ± 1.30 and weighing 400 kg were selected to monitor ovarian dynamics on alternate days by the same sonographer from the onset of heat through ovulation until the next ovulation. Experiment 2 measured the effect of estrus synchronization methods, controlled internal drug release-gonadotropin-releasing hormone (CIDR-GnRH, n = 31) and Ovsynch (OVS, n = 32) on various reproductive parameters. The mean estrous cycle length was 19.81 ± 0.56 days with two follicular waves. The mean inter-ovulatory interval was 20.80 ± 0.52 days, with a preovulatory follicular size of 13.83 ± 2.37 mm. Estrus response was higher (p > 0.05) in controlled internal drug release (CIDR)-GnRH (93.54%) than in OVS (84.37%) cows. Similarly, ovulation and conception rates were higher in CIDR-GnRH (91.66% vs. 68.42%) than in OVS cows (41.37% vs. 33.33%), respectively. In conclusion, CIDR-GnRH results in a better estrus response, higher ovulation rate, and subsequently greater conception rate than OVS in Bos indicus dairy cows.

Abstract Genital tumours are rare among cattle, largely due to their relatively short lifespans. Leio-myoma, a smooth muscle tumour being more prevalent in dogs, appears only at a rate of 1.00 - 2.00% in cattle, affecting reproductive efficiency in cases of complete uterine obstruction. This case report involves an 8-year-old cow with repeated insemination attempts unveiled 5.00 cm intra-luminal uterine mass, obstructing the right uterine horn. Transrectal sonography (TRUS) revealed a highly vascularized mass with normal ovarian function. Confirmation of clinical condition, i.e., uterine leiomyoma, via uterine biopsy concluded the presence of neoplastic smooth muscle cells arranged in interlacing bundles showing mild pleomorphism, and special staining using Masson’s trichrome revealed an unappreciable amount of connective tissue; subsequently right flank celiotomy was performed to remove the benign tumour. Forty-five days after celiotomy, TRUS examination confirmed an unobstructed uterine horn, and bilateral oviduct patency was adjudged with 2.50% methylene blue. Following treatment for chronic endometritis, artificial insemination led to conception nearly 90 days post-procedure. The TRUS aids preliminary diagnosis, while definitive identification demands necropsy and surgical methods. This case underscores the diagnostic significance of TRUS, histopathology and celiotomy for identifying and managing uterine leiomyoma in cattle.

Abstract The present research was carried out to assess the serum progesterone (P₄) concentration and uterine hemodynamics at estrus till ovulation in cyclic cows (N = 130) with healthy or diseased uterus. At estrus, 85 cows were diagnosed with clinical endometritis (CE; n = 44) and sub-clinical endometritis (SCE; n = 41); whereas, 45 cows being served as control namely no endometritis (NE; n = 45) were included in the study. Serum progesterone estimation at 12 - 14 and 40 hr after the onset of estrus and Doppler sonography of both middle uterine arteries were done to envisage the uterine hemodynamics and ovulation. The serum progesterone concentration was significantly higher at 12 - 14 hr after onset of estrus in CE and SCE cows. At 12 - 14 hr after onset of estrus, a cut-off value of ≥ 0.48 ng mL^-1 P₄ was obtained, above which 22.72% CE, 26.82% SCE and only 8.88% NE cows failed to ovulate within 36 - 40 hr of estrus onset. Among the Doppler indices, pulsatility and resistance indices were significantly higher; whereas, volume and velocity indices were significantly lower in NE cows. In cows diagnosed with CE and SCE, a higher supra-basal P₄ concentration, and velocity and volume of blood flow to uterus at estrus negatively affected the duration to ovulation.

Abstract This study designed a protocol that would combine pregnant mare serum gonadotrophin (PMSG) and cabergoline (CAB) to induce estrus in bitches. Twenty clinically healthy adult and anestrous female dogs were randomly assigned into four groups. The first group was treated with 5.00 μg kg^-1 CAB until the onset of proestrus or for 25 days. The second group was treated with 20.00 IU kg^-1 PMSG for 5 days and 500 IU human chorionic gonadotropin (hCG) on the 5^th day. The third group was treated with 5.00 μg kg^-1 CAB for 10 days in combination with 20.00 IU kg^-1 PMSG for 5 days and 500 IU hCG on the 10^th day. The control group received 1.00 mL of normal saline. Ovarian changes were evaluated ultrasonographically, and the estrus cycle phase was examined by vaginal cytology. Respectively, three, three and four bitches showed clinical signs of proestrus in each treatment group. The intervals between treatment and proestrus for each group were 30.00 ± 3.05, 7.67 ± 1.20 and 13.00 ± 1.20 days, respectively. Two weeks after estrus, the progesterone mean was 14.51 ± 6.24, 19.96 ± 17.16 and 19.12 ± 9.26 ng mL^-1 for each group, respectively. In ultrasonography examination, the largest follicle was identified at 15.66 ± 1.33, 11.66 ± 2.40 and 8.75 ± 2.17 days after the onset of proestrus and the largest follicle’s size was measured 6.50 ± 0.55, 4.83 ± 1.64 and 7.07 ± 1.49 mm for each group, respectively. Although the combined use of CAB and PMSG reduced the duration of treatment, alteration of the duration or PMSG dosage can be helpful to improve the results.

Abstract Although cryopreservation of ovarian tissue has advanced greatly, it remains a challenge, and protocols should be optimized to handle the heterogeneous nature of ovarian samples. In an effort to address this factor, the present study evaluated the effects of corpus luteum (CL) and side of ovaries (right versus left) on cellular morphology and viability of vitrified bovine ovarian fragments in a closed system. The ovaries were categorized according to whether they had a CL and which side they were on, and then divided into six groups: 1) CL+ (with CL) group; 2) CL˗ (without CL) group; 3) right ovaries group; 4) left ovaries group; 5) fresh control group (ovaries without vitrification or culture that were not selected for CL or ovarian side) and 6) In vitro culture medium control group (non-vitrified ovaries that were not selected for the presence or absence of CL or side of the ovaries). The current study shows that the CL˗ and right groups had the greatest percentage of follicles with normal morphology compared to other vitrified-warmed groups. Furthermore, the levels of necrosis and tissue damage of the right cultured group were the lowest compared to other groups. It was shown that bovine ovarian tissues derived from right ovaries and ovaries without a corpus luteum can be functionally and morphologically preserved after vitrification. For the first time, the present study suggests that bovine ovarian tissue vitrification can be improved by considering the origin of the ovaries.

Abstract About a third of human infertility is related to male factors. Of these, idiopathic-related infertility is not curable. Diabetes mellitus is a metabolic disorder affecting male impotence and fertility by increased production of free radicals and oxidative stress. Saponin, a glycosidic compound found in many plants, improves sperm parameters. The present study investigated the effect of saponin on sperm oxidative stress and testicular structure in streptozotocin (STZ)-induced diabetic mice. The diabetes was induced by the administration of 150 mg kg^-1 STZ via a single intra-peritoneal injection. All experimental mice were allocated to the following groups: Control group, diabetic control group, diabetic group administrated 100 mg kg^-1 saponin daily and one healthy group administrated saponin daily for 56 days. At the end of the treatment period, serum levels of insulin, glucose and oxidative stress markers were measured. A histological evaluation of testicles was performed. Treatment of diabetic mice with saponin ameliorated testicular tissue damage as well as serum glucose and insulin concentrations. Furthermore, in the diabetic group, the serum concentration of malondialdehyde was increased; while, the activity of superoxide dismutase and glutathione peroxidase enzymes was reduced. The mean Johnsen's score and the diameter and thickness of seminiferous tubules were lower in the diabetic mice than control ones. However, these parameters were higher in the saponin-treated mice than controls. Overall, saponin administration rectified all examined parameters. The anti-oxidant role of saponin improves sperm parameters and diabetes-induced testicular oxidative damage.

Abstract Triple-negative breast cancer (TNBC) is an aggressive and deadly breast cancer sub-type with limited therapeutic options. Dandelion (Taraxacum officinale) exhibiting extensive anti-cancer activity is reported to be effective against TNBC; however, its anti-tumor effect mechanisms have not been fully elucidated. The purpose of this study was to determine the anti-cancer activity of hydroalcoholic extract of dandelion (HADE) on 4T1 cells, and the mechanism of HADE-induced cell death. The effect of HADE on cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays. Apoptotic cell death was monitored by flow cytometry. The DNA fragmentation was evaluated by Acridine orange/Ethidium bromide (AO/EB) staining. Nitric oxide (NO) level was detected using Griess assay. The effects of HADE on Atg-7, Beclin-1, Bcl2, Bax and p53 genes were investigated by real-time reverse transcription-polymerase chain reaction. The results showed that HADE inhibited cell growth and proliferation in a dose- and time-dependent manner. The HADE induced 4T1 breast cancer cell death via apoptosis and autophagy. The DNA fragmentation was improved as the concentration of HADE increased. The NO secretion was declined with increasing concentration of HADE. Gene expression analysis confirmed HADE-induced apoptosis and autophagy in cancer cells. The Bax, Bax/Bcl-2 ratio, p53, Beclin-1 and Atg-7 over-expression as well as Bcl-2 down-regulation were also evident in treated cancer cells.

Abstract The purpose was to identify differentially expressed plasma microRNAs (miRNAs) in cows with clinical and subclinical endometritis. In this study clinical endometritis (CE; n = 23) based on vaginal discharge score (VDS), subclinical endometritis (SCE; n = 17) based on VDS (0), and endometrial cytology (the presence of 8.00% polymorphonuclear neutrophils (PMN) on days 21-31 and 5.00% on days 41-51 days in milk (DIM) and healthy cows (n = 21) based on vaginal discharge score (0), and endometrial cytology (< 5.00% PMN on days 21 - 31 and < 5.00% on days 41 - 51 DIM) were selected. The results showed that the expression level of miR-146a was significantly higher in the CE (19.17-fold), and SCE (6.22-fold) groups than those of healthy cows. The relative transcript abundance of miR-223 was considerably down-regulated in the CE (0.26-fold) and SCE (0.06-fold) compared to the healthy cows. The expression levels of miR-146a and miR-223 were significantly higher in the CE group which could be caused by Gram-negative bacterial infection. Our results showed that the expression level of plasma miRNAs postpartum could be used as a reliable marker to distinguish between SCE, CE and healthy cows.

Abstract Sperm survives for a very short time in fresh semen, and slow cooling to 5.00 ˚C kills a large number of sperms. This study was aimed to compare the semen quality parameters and anti-oxidant levels in four extenders (manual, Triladyl, Steridyl and AndroMed). Semen samples were obtained from a total number of 12 dual-purpose Simmental bulls kept in the Simmental Cattle Breeding Center for a period of 3 months using an artificial vagina. Sperm viability, motility, abnormal morphology, plasma membrane integrity, DNA damage, chromatin quality, total antioxidant capacity (TAC) and lipid peroxidation were evaluated. The highest progressive motility, viability, plasma membrane integrity, and TAC and the lowest levels of malondi-aldehyde in the frozen-thawed semen belonged to the semen group frozen with Triladyl. Parameters of motility were higher in the frozen-thawed semen with Triladyl than in other groups, indicating a significant difference from the manual extender. Among the extenders studied, Triladyl was the most suitable for semen freezing in Simmental bulls.

Abstract The creole breed Blanco Orejinegro (Bos taurus adapted) is an important animal genetic resource in Colombia. However, very little is known about the reproductive physiology of the breed. The objective was to determine the dynamics of progesterone circulation and follicular growth in Blanco Orejinegro cows throughout the estrous cycle. Fifteen cyclic Blanco Orejinegro cows were used and subjected to hormonal protocol for estrous and ovulation synchronization. Once the time of ovulation was identified by monitoring the ovarian dynamics with ultrasonography equipment. For description of the results, the mean was used as a measure of central tendency and the standard error of the mean. Statistical analysis was longitudinal descriptive. Blood samples were collected every 24 hr throughout the estrous cycle for serum quantification of progesterone (P4), monitoring of corpus luteum (CL) growth dynamics and follicular dynamics. It was possible to verify that 76.92% of the Blanco Orejinegro cows presented two follicular waves during the estrous cycle and the highest circulating levels of P4 (> 6.00 ng mL^-1) were observed on the 14^th day of the estrous cycle. It was concluded that the creole cows of the Blanco Orejinegro breed presented two follicular waves per estrous cycle. In addition to, the size of the CL was consistent with the secretion of progesterone presenting higher circulating levels at the end of the luteal phase.