The protease inhibitor antipain has a beneficial synergistic effect with trehalose for ram semen cryopreservation.

The objective of this study was to examine the different concentrations of antipain and trehalose combination on post-thawed quality of ram semen cryopreserved in tris extender. Ejaculates were collected from four rams using the artificial vagina, pooled at 37°C and diluted with (A0  Tre0 : antipain 0 µM and trehalose 0 mM (Control); A10  Tre0 ; A50  Tre0 ; A0  Tre30 ; A0  Tre60 ; A10  Tre60 ; A10  Tre30 ; A50  Tre30 and A50  Tre60 ). Diluted semen samples were gradually cooled down from 37 to 5°C in a cold cabinet; then, they were loaded into 0.25 ml straws, frozen and stored in liquid nitrogen. Sperm motility (CASA), viability, membrane functionality and abnormality were evaluated after thawing process. Progressive motility in extender supplemented with A10  Tre0 , A0  Tre30 and A10  Tre60 significantly (p < 0.05) higher as compared to the control (A10  Tre0 ). A10  Tre60 (47.50 ± 0.73) provided the best maintenance of progressive motility in comparison with the control (40.50 ± 0.73). No significant differences were observed between all treated groups in terms of total motility, VAP, VSL, VCL, ALH, BCF, STR and LIN. The percentages of sperm with viable were significantly higher in extenders supplemented with A10  Tre0 , A50  Tre0 , A0  Tre30 and A10  Tre60 , compared to control. Addition of A10  Tre0 , A50  Tre0 and A10  Tre60 to extenders improved the percentages of sperm abnormality, compared to the controls. A10  Tre60 (67.84 ± 1.51) treatment provided the best maintenance of normal morphology compared to the other treatments. The supplementation with A10  Tre0 , A0  Tre60 and A10  Tre60 improved the percentage of sperm membrane functionality when compared to the control (p < 0.05). Comparing these results with those of control diluents, the effects of supplementation were better except for A50  Tre60 group. In conclusion, when combination of antipain (10 µM) and trehalose (30 and 60 mM) was added, they conferred a great cryosurvival capacity with their synergic effects during freeze-thawing process.

Effect of Exogenous Progesterone or Flunixin Meglumine After AI on Serum Progesterone Concentration and Pregnancy per AI in Lactating Dairy Cows.

The objective of this study was to determine the effect of post AI administration of exogenous progesterone (P4) or a prostaglandin F2α (PGF2α) synthesis inhibitor agent on serum P4 concentrations and pregnancy per AI (P/AI) in lactating dairy cows. Eighty lactating cows were randomly allocated to one of four treatment groups: 1) CON (control), received 5 mL of saline solution on d 6 and 14 post AI; 2) IP4 (injection of P 4), received 125 mg of P4 im on d 6 and 14 post AI; 3) CIDR, received a controlled internal drug release insert containing 1.38g of P4 from d 6 to 20 post AI; and 4) FM (Flunixin Meglumine), received 0.625 g of Flunixin Meglumine, a nonsteroidal anti-inflammatory drug, twice daily on d 15 and 19 post AI. Blood samples were taken on d 0, 6, 14, 17 and 20 post AI to determine P4 concentrations. Transrectal palpation was performed between 40 and 45 d post AI to determine pregnancy status. All treatment groups (i.e. IP4, CIDR and FM) resulted in greater serum P4 concentration on d 17 and 20 post AI compared to CON (P < 0.05). Cows given a CIDR insert had greater concentrations of P 4 on d 17 and 20 than IP4 and FM cows (P < 0.05). However, no significant difference was found between IP4 and FM groups for serum P4 concentrations. The P/AI was greater (P < 0.05) in CIDR-treated cows (55%, 11/20) than CON (25%, 5/20), and intermediate in IP4 (40%, 8/20) and FM (35%, 7/20) cows. In summary, treatment with exogenous P4 (i.e. CIDR and IP4) or FM increased serum P4 concentrations in lactating dairy cows. However, results suggest that only CIDR administration would improve P/AI.

The presence of corpus luteum may have a negative impact on in vitro developmental competency of bovine oocytes.

The aim of the current study was to investigate the effects of the presence or absence of corpus luteum (CL) on in vitro developmental competence of bovine oocytes. In experiment 1, cumulus oocyte complexes (COCs) were collected from slaughterhouse ovaries and divided according to the presence (CL(+) oocytes) or absence (CL(-) oocytes) of a CL in the ovary. Control oocytes (C group) were obtained from ovaries which were not selected toward the presence or absence of CL. All oocytes were submitted to in vitro maturation, fertilization and culture. In experiment 2, the oocytes from the CL(+) and CL(-) ovaries were divided into grown (BCB(+)) and growing (BCB(-)) categories by means of the brilliant cresyl blue (BCB) test. The oocytes from all groups (CL(+)/BCB(+), CL(-)/BCB(+), CL(+)/BCB(-), CL(-)/BCB(-) and control oocytes) were subjected to in vitro embryo production. In experiment 1, the cleavage and blastocyst rates of CL(-) oocytes were higher than those of CL(+) oocytes (83.9% and 43% vs. 69.3% and 22.5%, respectively). In experiment 2, there was less BCB(+) oocytes (more competent oocytes) in the group of CL(+) oocytes than in the group of CL(-) oocytes. Furthermore, developmental competence of all CL(+) oocytes (CL(+)/BCB(+) and CL(+)/BCB(-)) was lower than that of all CL(-) oocytes (CL(-)/BCB(+) and CL(-)/BCB(-)). Thus, the presence of a corpus luteum in the ovary may have negative effects on developmental competence of ipsilateral oocytes.

In vitro developmental competence of bovine oocytes: Effect of corpus luteum and follicle size.

BACKGROUND: Previous studies reported many discrepancies about the effects of corpus luteum (CL) and ovarian follicle size on the developmental competence of oocytes. OBJECTIVE: The aim of this study was to investigate the effects of CL and different size of follicle on the developmental potential of bovine oocytes. MATERIALS AND METHODS: After ovarian classification based on presence or absence of CL, sample follicles were placed in three groups according to their diameter; small (S; 3-6 mm), medium (M; 6-9 mm), and large (L; 10-20 mm). Collected oocytes in each group were subjected to the in vitro embryo production processes. RESULTS: Results showed that, the percentages of blastocyst obtained from oocytes originating from small and medium follicles of ovaries bearing a CL (CL+S-oocytes and CL+M-oocytes, respectively) were lower (p<0.001) than those of small and medium follicles of ovaries not bearing a CL (CL-S-oocytes and CL-M-oocytes, respectively) (30.8% and 33.6% vs. 36.9% and 38.7% respectively). Although, the percentages of blastocyst obtained from CL-M-oocytes and CL-L-oocytes were greater (p< 0.001) than those of CL+S-oocytes and CL+M-oocytes. There were no significant differences in the percentages of blastocyst formation between controls (C-oocytes), CL-S-oocytes and CL+L-oocytes. CONCLUSION: According to the results of this study, the negative effect of CL on the developmental competence of bovine oocyte depends on the follicle size. Therefore, oocytes originating from large grown follicles were not influenced by negative effects of CL as much as those originating from small and medium follicles did.

Comparing the effect of oral supplementation of vitamin E, injective vitamin E and selenium or both during late pregnancy on production and reproductive performance and immune function of dairy cows and calves.

The object of this study was to determine the effect of prepartum supplementation of vitamin E with or without injective vitamin E and selenium (Se) on productive and reproductive performances and immune function in dairy cows. Sixty multiparous Holstein dairy cows were divided randomly into three groups at the end of gestation. Cows in each group received one of three treatments: (1) a single intramuscular (im) injection of vit. E + selenium 3 weeks prepartum; (2) daily supplementation of oral vit. E given from 3 weeks prepartum to parturition; (3) injective vit. E + Se with daily supplementation of oral vit. E. Blood samples were collected from cows at calving and from calves at 0 and 7 days of age. Concentration of IgG in serum of cows and calves as well as in colostrum was determined. No significant differences among treatments occurred in the concentrations of IgG, animal, and calf production and reproduction performance. Due to the lack of significant difference between injection and oral supplementation, it is recommended to replace the injection with oral supplementation.

Developmental competence of bovine oocytes selected based on follicle size and using the brilliant cresyl blue (BCB) test.

BACKGROUND: Many studies reported that follicle size has an essential role in developmental potential of oocytes. Also, the brilliant cresyl blue (BCB) test is one of the most important criteria in selection of more competent oocytes. OBJECTIVE: Selection of developmentally competent bovine oocytes. MATERIALS AND METHODS: A total of 1730 bovine cumulus oocyte complexes (COCs) were recovered from the ovaries by follicles isolation and classified into 3 categories according to the diameters of the follicles (small, <3 mm; medium 3-6 mm and large >6 mm). Oocytes were exposed to the BCB stain, diluted in Dulbecco's phosphate-buffered saline, modified with 0.4% bovine serum albumin (BSA) for 90 min. Oocytes with or without blue coloration of the cytoplasm were designated as BCB(+) and BCB(-), respectively. RESULTS: The BCB(+) and control oocytes originated from large and medium follicles exhibited a higher (p<0.0001) cleavage and blastocyst rate than BCB(-) oocytes. Furthermore, the BCB(+) oocytes from large and medium follicles had the highest (p<0.0001) proportion of blastocyst than other treatment groups. In contrast, the BCB(-) oocytes from small follicles had the lowest (p<0.0001) proportion of blastocyst than other treatment groups. Interestingly, the percentage of the BCB(+) oocytes from the large and medium ovarian follicles was significantly higher (p<0.0001), than the BCB(+) oocytes from the small follicles. CONCLUSION: Current results confirmed that each BCB(+) oocyte could not lead to perfect embryo development and the BCB test is not sufficient enough for the identification of oocytes that are competent for in vitro embryo development.

Effect of various concentrations of Minimal Essential Medium vitamins (MEM vitamins) on development of sheep oocytes during in-vitro maturation.

BACKGROUND: Improvements in culture media formulations have led to an increase in the ability of sheep embryo in culture throughout the preimplantation period. OBJECTIVE: This study was carried out to evaluate the effects of various concentrations of MEM vitamins during in vitro maturation of sheep oocytes and subsequent embryo development. MATERIALS AND METHODS: Sheep ovaries were collected from a slaughterhouse and transported to the laboratory. Oocytes were matured in SOF medium supplemented with, eCG, hCG and EGF in various concentrations of MEM vitamins (control, 0.5, 1 and 1.5 ) for 24h. The cumulus oocyte compelex (COCs) were co-incubated with epididymal spermatozoa of post mortem rams in synthetic oviduct fluid fertilization (SOFF) medium with 10% heat inactivated estrous sheep serum for 18h. Embryos were cultured in synthetic oviduct fluid culture 1 (SOFC1) medium for 48h followed by cultured in synthetic oviduct fluid culture 2 (SOFC2) medium for six days. RESULTS: Addition of 0.5 and 1   MEM vitamins significantly increased (P< 0.05) overall blastocyst development (21.62% and 22.33%; respectively) compared with 1.5 MEM vitamins (15.59%), but there was no difference between control, 0.5 and 1 MEM vitamins in the percentage of embryos successfully developing to the blastocyst stage (19.50%, 21.62% and 22.33% respectively). CONCLUSION: It seems that addition of 1.5  of MEM vitamins has detrimental effect on blastocyst rate.

The effect of various concentrations of myo-inositol in culture medium on development of bovine embryos.

BACKGROUND: Progress achieved in culture media formulations have resulted led to an improvement in maintaining the mammalian embryo in culture throughout the preimplantation and pre-attachment period. OBJECTIVE: The objective of this study was to evaluate the effect of various concentrations of myo-inositol during in vitro fertilization of bovine oocytes on subsequent embryo development. MATERIALS AND METHODS: Bovine cumulus oocytes complexes (COCs) were matured in vitro at 39(o)C, in humidified 5% CO2 atmosphere for 22-24 h. The COCs were co-incubated with epididymal spermatozoa of post mortem bulls in modified TALP medium for 22-24hr. The fertilization medium used was: 1) TALP medium without myo-inositol (control); 2) control+0.02 g/l myo-inositol; 3) control+0.03 g/l myo-inositol; 4) control+0.04 g/l myo-inositol. Zygotes were cultured in vitro for 8 days when the ratios of in vitro embryo development of the hatched blastocysts were assessed and compared with the control group (p<0.05). RESULTS: The presence of 0.04 g/l myo-inpsitol significantly improved overall morula and blastocyst rates (46.94%) compared to control (32.19%), but there was no difference in the percentage of embryos successfully developed to the morula and blastocyst stage when different levels of myo-inositol were used (46.94, 36.36 and 37.33% respectively). The mean percentage of cleavage rate was not significantly affected by treatments. CONCLUSION: These results suggest that, addition of 0.04 g/l myo-inositol in TALP medium is more beneficial for subsequent bovine embryonic development.