The fertility of dairy heifers and cows is not influenced by the follicular wave of the ovulatory follicle.

Two studies were conducted to evaluate the effects of the follicular wave on ovarian function and fertility in dairy heifers and lactating cows. In study 1, the estrous cycle of the selected Holstein heifers was initially synchronized using two intra-muscular prostaglandin F2α (PGF2α) administrations 11 days apart. Heifers in group FFW (n = 14) received an intra-muscular 500 µg PGF2α administration on day 7 after detecting standing estrus, while Heifers in group SFW (n = 14) were administered PGF2α 13 days after detecting standing estrus. The pregnancy rates of FFW (n = 98) and SFW (n = 100) heifers were also determined 35-37 days after artificial insemination (AI). In Study 2, healthy Holstein lactating cows (n = 28) were randomly assigned to either the FFW (n = 14) or SFW (n = 14) groups. The estrous cycles of the cows were presynchronized using two intra-muscular administrations of PGF2α given 14 days apart. Then, the emergences of the follicular waves were induced using an Ovsynch protocol. The pregnancy rate of FFW (n = 99) versus SFW (n = 98) cows was also determined 35-37 days after AI. The ovulatory follicle and corpus luteum (CL) resulting from the ovulatory follicle of FFW were larger than those of the dominant follicle and the CL of SFW in dairy heifers and lactating cows. However, the pregnancy rate did not differ between the FFW and SFW groups in heifers and lactating cows 35-37 days after AI. In conclusion, although the characteristics of the ovulatory follicles in FFW versus SFW animals differed, the follicular wave in dairy heifers or lactating cows did not affect fertility.

Supplementation of melatonin to cooling and freezing extenders improves canine spermatozoa quality measures.

BACKGROUND: Sperm freezing and cold storage are the two most common assisted reproductive technologies in the canine breeding industry. The freeze-thawing process causes significant detrimental changes in both sperm cell structure and function. Previous research has confirmed that excessive accumulation of un-scavenged free radicals (oxidative stress) plays an important role in the cryopreservation-induced damage to sperm cells. Also, the gradual accumulation of the free radicals during cold storage leads to a decline in the sperm quality markers. Melatonin is an endogenous neurohormone synthesized from tryptophan amino acid by pineal glands. Besides its several well-known physiologic roles, melatonin has a significant antioxidant potential through direct free radical scavenging properties. Therefore, the current study was designed to evaluate the potential in vitro protective properties of melatonin (0.5, 1, and 2 mM) on canine sperm cells after freezing or during long-term cold storage (9 days, 5 °C) on most important sperm in vitro fertility markers. RESULTS: Melatonin at 0.5, 1- or 2-mM concentrations could preserve significantly higher sperm total motility after 4 days of cold storage. However, only the 1- and 2 mM melatonin concentrations could result in better TM and PM values after 7 days of cold storage. Furthermore, melatonin supplementation could preserve higher sperm viability and acrosome integrity after 7 days of storage. Also, it could have significant protective effects on the cooled sperm DNA integrity. In the freezing section of the current research, melatonin at either 1- or 2-mM concentrations could not improve the sperm post-thaw TM and PM, whereas they improved sperm DNA integrity. Also, the post-thaw plasma membrane functional integrity and sperm velocity parameters were not affected by the treatment. Although DMSO (Dimethyl Sulfoxide) as the melatonin solvent could reduce the level of sperm lipid peroxidation and even improve the post-thaw sperm DNA integrity compared to the negative control, it reduced the post-thaw sperm progressive motility. However, the negative effects were reversed by concurrent melatonin supplementation at 1- and 2-mM concentrations. CONCLUSION: The addition of 1- or 2-mM melatonin to the canine sperm freezing and cooling media could improve sperm motility, viability, acrosome, and DNA integrity.

Frozen-thawed ampullary cell monolayer improves bovine embryo in vitro development and quality.

The aim of this study was to evaluate the effects of different timing for frozen-thawed bovine ampullary epithelial cell (BAEC) and bovine oviductal epithelial cell (BOEC) co-culture on the development and quality of bovine embryos produced in vitro. Embryo development was assessed by day 8 blastocyst yield, whereas embryo quality was determined using blastocyst differential cell count, cryotolerance and the expression of selected genes related to embryo quality. The results showed that the presence of BAECs during the last 6 h of in vitro maturation (IVM) increased blastocyst yield and survival of the vitrified-warmed blastocysts. In addition, embryos produced in the presence of BAECs during the last 6 h of IVM or in the presence of BOECs during the first 4 days of in vitro culture (IVC) showed a greater number of trophectoderm cells and a greater inner cell mass. In terms of gene expression, IFN-T was downregulated and PLAC8, AQP3 and ATP1A1 were upregulated in the presence of the BAECs during the last 6 h of the IVM and/or in the presence of BOECs during the first 4 days of IVC. In conclusion, co-culturing bovine oocytes with a frozen-thawed ampullary cell monolayer during the last 6 h of maturation increased blastocyst yield and quality.

Bovine salpingitis: Histopathology, bacteriology, cytology and transcriptomic approaches and its impact on the oocyte competence.

The present study was performed to examine the histopathology, cytology, bacteriology and expression pattern of a targeted set of genes of cytokines in the oviduct of cows with inflammation (Experiment 1). In addition, the effects of oviductal fluid from cows with salpingitis on the oocyte maturation and fertilization in vitro were examined (Experiment 2). The most frequent bacterial co-infection was Escherichia coli and Fusobacterium necrophorum, which was always associated with severe histopathologic salpingitis. Out of 15 cows with histologically healthy uterus, only one cow (6.7%) displayed the histologic signs of mild salpingitis, whereas from 50 cows with endometritis, 48 cows (96%) showed histologically different grades of salpingitis. The mRNA expression of IL1ß, CD14, IL8 and CASP3 was significantly different among all groups of salpingitis (P < 0.05) with the highest level of mRNA expression in the sever grade of salpingitis. Results of experiment 2 showed a significant decline in the oocytes with peripheral free mitochondria and fertilization rate in the salpingitis group than the no- salpingitis group (P < 0.05). In conclusion, our results showed that histologically detected salpingitis is in most cases associated with histologic and cytologic endometritis. The pattern of the gene expression of chemokines and cytokines was altered in association with different grades of salpingitis. Further, we observed a decline in the peripherally located mitochondria and lower fertilization rate in oocytes following addition of oviductal fluid collected from the cows with sapingitis to the maturation media.

Intrauterine infusion of blood serum of dromedary camel improves the uterine health and fertility in high producing dairy cows with subclinical endometritis.

The blood serum of dromedary camels contains a unique type of antibodies with a high potency to neutralize toxins and to identify and inactivate some bacterial pathogens. The present study was designed to examine changes in the endometrial histology of cows with no subclinical endometritis (SE) (experiment 1) and changes in the uterine cytology and endometrial mRNA expression of COX2, IL-1ß, IL-8, and iNOS following intrauterine administration of DCBS in cows with SE as compared to different common treatments (experiment 2). In addition, the effects of the intrauterine administration of DCBS were examined on the pregnancy rate in dairy cows with SE (experiment 3). DCBS did not induce any histological reactions in the bovine endometrium. The mean ( ± SE) percentage of PMNs after intrauterine infusion of Pen-Strep, DCBS and double DCBS in cows with SE differed as compared to cows treated with PGF2α and no treated cows with SE (1.47 ± 0.87; 1.43 ± 1.08 and 1.31 ± 0.23 vs 3.00 ± 0.43 and 3.5 ± 0.75, P < 0.05, respectively) in experiment 2. The mRNA expression of COX2, IL-1ß, and iNOS was reduced (P < 0.05) after treatment with Pen-Strep, DCBS and double DCBS as compared with no treated-cows with SE. The pregnancy rate after the first AI was tended to be higher (49.2 vs 39.0%), while the overall pregnancy rate was greater (P < 0.05) in cows with SE when treated with DCBS as compared to the Pen-Strep group (76.9 vs 61.0%) in experiment 3. In conclusion, serum of dromedary camel, as a non-antibiotic preparation, can improve the uterine health and fertility when used for the treatment of bovine SE.

Bovine oocyte developmental competence and gene expression following co-culturing with ampullary cells: An experimental study.

BACKGROUND: There is no sufficient information on the impact of bovine ampullary oviductal epithelial cells (BAOECs) on in vitro oocyte maturation competence and gene expression. OBJECTIVE: This study aimed to examine the oocyte developmental competence following co-culturing with a monolayer of fresh and frozen-thawed ampullary cells. MATERIALS AND METHODS: Bovine cumulus-oocyte complexes (COCs) were distributed into three groups: control group; where in COCs were cultured in cell-free media for 24 hr and FML and FTML groups in which the COCs were cultured in maturation media for 18 hr and then transferred into a media containing fresh and frozen-thawed BAOECs monolayer, respectively (BAOECs were extracted from the oviducts of slaughtered cattle and were then cultured freshly or frozen-thawed) for a further 6 hr. After 24 hr, the expanded COCs were evaluated for nuclear maturation, fertilization rate, and gene expression (GDF9, StAR, CASP3, and FSHr). RESULTS: Nuclear maturation rate in the FTML group was significantly higher than the control group (p = 0.02). The fertilization rate of FTML group was significantly higher than the control and FML groups (p = 0.05 and p = 0.03, respectively). In terms of gene expression, GDF9 were upregulated in the presence of the BAOECs during the last 6 hr of the in vitro maturation (p < 0.001). Furthermore, the expression of the StAR gene in the FTML group was higher than the other groups (p = 0.02). CONCLUSION: Ampullary cells co-culturing (especially frozen-thawed cells) for in vitro maturation of bovine oocytes yields encourages the results and demonstrates the beneficial effect of co-culture on gene expression and developmental competence.

Effects of Pre-ovulatory Follicular Fluid of Repeat Breeder Dairy Cows on Bovine Fertility Transcriptomic Markers and Oocytes Maturation and Fertilization Capacity.

The current study aimed to determine the effects of the preovulatory follicular fluid (FF) of normal heifer (NH) and repeat breeder cows with subclinical endometritis (SCE) or without (nSCE) on oocyte maturation (Experiment 1) and fertilization rates (Experiment 2). Moreover, the pattern of gene expression of cumulus oocyte-complexes was evaluated in Experiment 1. In Experiment 1, nuclear maturation in the nSCE group was higher, compared to that in the SCE group (P = 0.05). In addition, the oocyte nuclear maturation in the normal heifer was significantly higher, in comparison to that of SCE groups (P < 0.05). Furthermore, the mean percentage of normal oocyte fertilization was higher in the nSCE group, compared to that in the SCE group (P < 0.05). The expressions of growth differentiation factor, GDF9; steroidogenic acute regulatory, StAR and follicle-stimulating hormone receptor, FSHr in the NH group were significantly higher, compared to those in SCE and nSCE groups (P < 0.05). Moreover, the expressions of all genes in the nSCE group were not significant, in comparison to those in the SCE group (P > 0.05). The supplementation of oocyte maturation medium with FF from pre-ovulatory follicles of repeat breeder cows resulted in less oocyte maturation and cumulus cell expansion. In conclusion, the lower fertility in RB cows could be ascribed to the lower oocyte maturation rate and less expression of GDF9, StAR, and FSHr in the cumulus-oocyte complexes.

Nicotinic Acid (Niacin) Supplementation in Cooling and Freezing Extenders Enhances Stallion Semen Characteristics.

In the present study, we aimed to evaluate the possible protective effects of the nicotinic acid (NA) at three concentrations (10, 20, and 40 mM) on the equine cooled and frozen-thawed spermatozoa quality markers including viability, plasma membrane or acrosome integrity, DNA fragmentation, lipid peroxidation, and total oxidant levels. We also evaluated the effects of NA on preservation of the post-thaw sperm quality after 6 hours of cold storage before freezing. Five stallions were used for semen collections. The current experiment was repeated six times using pooled semen samples from two stallions, each time. We showed that NA at 20 and 40 mM concentrations could significantly improve the stallion sperm quality markers during cold storage. However, the protective effects were not different between 20 mM and 40 mM concentrations in most measures. Nicotinic acid could also improve the post-thaw stallion sperm quality at 10, 20, and 40 mM concentrations. However, the 40 mM concentration showed a negative impact on some post-thaw kinematic sperm parameters. Nicotinic acid at 10 and 20 mM concentrations could preserve the sperm cryo-tolerance to be frozen up to 8 hours after collection without a significant decline in most of the post-thaw sperm quality measures. Nicotinic acid could also decrease the level of the lipid peroxidation and total reactive oxygen/nitrogen species in the cooled and frozen-thawed spermatozoa, in a dose-dependent manner. Therefore, NA at 20 mM concentration could preserve most of the stallion sperm quality measures during cold storage (42 hours, 5°C) and enabled storage of cooled stallion semen for 6 hours before freezing without significant deterioration of the post-thaw sperm quality.

Niacin improves maturation and cryo-tolerance of bovine in vitro matured oocytes: An experimental study.

BACKGROUND: Nicotinic acid (niacin) is a broad-spectrum lipid-modifying agent that has potent antioxidant properties and reduces the production of lipid peroxidation. OBJECTIVE: The purpose of the present study was to investigate the maturation, embryo development and cryo-tolerance merit, and levels of malondialdehyde (MDA), total oxidant status, and total antioxidant capacity following the supplementation of bovine oocytes maturation medium with different concentrations of niacin. MATERIALS AND METHODS: Immature cumulus-oocyte complexes were cultured in tissue culture medium-199 maturation media supplemented with 0, 100, 200, and 400 µM niacin under a standard in vitro culture condition. After 24 hr of culture, the nuclear maturation rate was assessed. Then, two groups of immature cumulus-oocyte complexes were cultured in TCM-199 either with or without 400 µM niacin and evaluated for embryo development. Also, matured cumulus-oocyte complexes in both groups were frozen using a standard vitrification procedure. After vitrification, oocytes were warmed in two steps and evaluated for embryo development. In addition, the level of total antioxidant capacity, total oxidant status, and MDA were measured. RESULTS: The results indicated that although the treatment with 400 µM niacin increased in vitro nuclear maturation (87.6±5.3), it did not improved the embryo development to the blastocyst stage. Higher cleavage and blastocyst rates were observed in vitrified oocytes that were cultured with supplemented 400 µM niacin compared to the control group (without niacin) (53.6±2.7 and 10.6±1.6 vs. 46.2±4.1 and 6.3±2.4, respectively). Also, the addition of 400 µM niacin to the maturation media could decrease MDA levels after maturation. CONCLUSION: Niacin could improve the quality of in vitro embryo production (IVP) embryos and tolerance of bovine oocytes to vitrification.

Oocyte maturation, embryo development and gene expression following two different methods of bovine cumulus-oocyte complexes vitrification.

OBJECTIVE: To examine the maturational competence, embryo development and expression of genes involved in oocyte maturation and cumulus expansion (GDF9, BMP15, HAS2, TNFAIP6, FGF17 and FSHr) following two standard methods of bovine COCs vitrification. METHODS: Bovine cumulus-oocyte complexes (COCs) were aspirated from slaughtered ovaries and then distributed into three groups: non-vitrified COCs (control), vitrification 1 group (V1); vitrification was performed by 15% ethylene glycol (EG) and 15% DMSO in holding media (TCM-199 with 20% FCS); and vitrification 2 group (V2); vitrification was performed by 40% EG in holding media. After vitrification, COCs were warmed in two steps and cultured and then evaluated for nuclear maturation, embryo development and gene expressions. RESULTS: The mean (±SD) percentages of nuclear maturation and blastocyst/cleaved were higher in control group (79.5 ± 8.0 and 31.0 ± 5.1%) than the V1 (34.8 ± 9.1 and 4.4 ± 5.1%) and V2 (47.8 ± 11.7 and 7.1 ± 5.8%) groups (P < 0.05), respectively. Further, COCs in V2 group showed higher mean (±SD) percentages of cleavage compared to V1 group (31.8 ± 1.0 vs 21.7 ± 2.8%; P < 0.05). GDF9 and BMP15 expression levels were higher in COCs in the control than of the vitrification groups (P < 0.05). In addition, expression level of GDF9 and BMP15 was higher in V2 group than in V1group (P < 0.05). The expression of HAS2 and FGF17 in V1 group was lower (P < 0.05) than that of the V2 groups. CONCLUSIONS: Expression of oocyte maturation genes was affected by vitrification procedure and conditions. Using EG alone for vitrification of bovine immature COCs, resulted in higher expression of GDF9, BMP15 and production of more in vitro matured and cleaved oocytes.

Protective effects of garlic aquous extract (Allium sativum), vitamin E, and N-acetylcysteine on reproductive quality of male rats exposed to lead.

The objective of present study was to investigate the effects of aqueous garlic extracts, vitamin E and N-acetylcysteine on lead-induced lipid peroxidation, changes in antioxidant defense system and semen quality in the rat testes. Twenty-five male rats were divided into five groups. Animals within different treatment groups were maintained on their respective diets for 35 days as follows: group 1 rats served as control and received water and standard pellets as food ad libitum; group 2 received lead acetate by gavage (1000 ppm); group 3 was treated with A. sativum extract (400 mg kg(-1), by gavage) plus lead acetate (1000 ppm); group 4 was treated orally with vitamin E (300 mg of alpha-tocopherol per kg of chow) plus lead acetate (1000 ppm); group 5 was treated orally with N-acetylcysteine (800 ppm)plus lead acetate (1000 ppm). The weights of testes, epididymis, epididymal sperm count, viable and motile sperms decreased significantly (p < 0.05) in lead-exposed rats. However treatment with vitamin E and aqueous garlic extract resulted in a significant (p < 0.05) increase in sperm motility and viability. Exposure to lead acetate significantly increased malondialdehyde (MDA) level with a significant decrease in the superoxide dismutase (SOD) activities in the testes of rats while co-administration of vitamin E and lead caused a significant (p < 0.05) decrease in MDA concentration compared with lead-exposed group. These results suggest that both vitamin E and in to a lesser extent aqueous garlic extract have a potent antioxidant protection in the testes of rat against the lead-induced oxidative stress.