Effects of ammonia and urea in vitro on mRNA of candidate bovine endometrial genes.

Large amounts of protein intake are associated with elevated ammonia and urea concentrations in both plasma and uterine fluid in dairy cows. These increased concentrations affect successful embryo development and subsequent pregnancy establishment. The objective of the present study was to examine the effects of ammonia and urea on the expression of some candidate genes in the endometrium of mid-luteal phase of the estrous cycle of dairy cows. Endometrial explants were cultured and treated with 0, 75, 150, 300, 600µM of ammonium chloride or 0, 4, 8, 12, 16mM of urea. After the RNA extraction and reverse transcription, real time PCR was performed to assess the treatment effects on relative amounts of mRNA of candidate genes. BCL2 mRNA was greater in explants treated with 150µM of ammonium chloride compared to explants treated with 0, 75 and 300µM. Relative amounts of IGFBP1 mRNA were less in explants treated with 600µM of ammonium chloride when compared with other concentrations. Relative FGF2 gene expression was less in explants treated with a greater concentration (600µM) of ammonium chloride or urea (16mM) when compared with lesser concentrations. Expression of HSPA1A, IGFBP3 and SERPINA14 genes was greater in explants exposed to lesser concentrations (150µM) of ammonium chloride or urea (4mM). Relative amounts of IGF1 and BAX mRNA were not affected by any of the ammonium chloride or urea concentrations tested. In conclusion, greater concentrations of ammonia and urea have negative effects on some endometrial gene expression, while moderate concentrations have positive effects.

mRNA of luteal genes associated with progesterone synthesis, maintenance, and apoptosis in dairy heifers and lactating dairy cows.

This study was performed to investigate the role of corpus luteum (CL) in reduced pregnancy rates (PR) observed in high producing lactating dairy cows. Development of CL and secretion of progesterone (P(4)) play a key role in early embryo development, implantation, and maintenance of pregnancy. Time of ovulation was synchronized in dairy heifers and second/third parity lactating dairy cows and CL enucleated surgically under local anesthesia on day 10 of the estrous cycle. Quality of the CL in dairy heifers (n=5) and lactating dairy cows (n=5) was compared by analyzing the expression of candidate genes by mRNA assessments using quantitative real-time PCR. Amounts of mRNA for factors associated with P(4) synthesis: 3betaHSD, anti-apoptotic function: BCL2, angiogenesis: VEGF, IGF1, and FGF2, and luteal maintenance: IL1A were greater (P<0.05) in CL obtained from dairy heifers compared to that of lactating dairy cows. Also a greater ratio for BAX:BCL2 mRNA was observed in lactating cows. Therefore, genes regulating angiogenic, steroidogenic, and luteotropic factors are highly expressed in heifers compared to lactating dairy cows, whereas apoptosis seemed to be more evident in CL of lactating cows. These findings suggest that CL of lactating dairy cows have reduced luteotropic as well as steroidogenic capacities on day 10 of the estrous cycle and might have played a critical role in reduced PR observed in lactating dairy cows.

Differential mRNA expression in in vivo produced pre-implantation embryos of dairy heifers and mature cows.

Decreasing fertility with increasing parity is considered to be a major constraint in the reproductive management of dairy cows. Even though pregnancy rates (PR) in mature cows have declined drastically in the last 50 years, it has remained constant in heifers. Early embryonic loss is a major cause for the loss of pregnancy in cows. Expression of developmentally important genes is vital for the function and survival of embryos. Hence, in this study, we compared the mRNA abundance of GLUT5, INFtau, HSP70, Na/K-ATPase, BAX, and BCL2 genes in the pre-implantation embryos of dairy heifers and mature cows. Heifers (n = 25) and cows (n = 20) were superovulated and artificially inseminated on the day of estrus. On day 7, the embryos were flushed and morphologically graded and RT-PCR was performed. HSP70 was expressed more in the grade I embryos in heifers than in cows, and in the grade I embryos of heifers than in grade II embryos of heifers. In pooled embryos (both grades I and II) of heifers and cows, expression for INFtau was greater in heifers than in cows. Grade I embryos had a higher expression of GLUT5 and Na/K-ATPase than the grade II embryos of cows. From this study, we conclude that there is differential expression of some developmentally important genes between embryos of heifers versus cows and between grades I and II embryos regardless of the embryo source. Future research will be necessary to elucidate any potential cause and effect between these genes and reduced PR observed in dairy cows.

Pregnancy rates to timed artificial insemination in dairy cows treated with gonadotropin-releasing hormone or porcine luteinizing hormone.

We compared the effects of porcine luteinizing hormone (pLH) versus gonadotropin-releasing hormone (GnRH) on ovulatory response and pregnancy rate after timed artificial insemination (TAI) in 605 lactating dairy cows. Cows (mean+/-SEM: 2.4+/-0.08 lactations, 109.0+/-2.5 d in milk, and 2.8+/-0.02 body condition score) at three locations were assigned to receive, in a 2x2 factorial design, either 100 microg GnRH or 25mg pLH im on Day 0, 500 microg cloprostenol (PGF) on Day 7, and GnRH or pLH on Day 9, with TAI 14 to 18h later. Ultrasonographic examinations were performed in a subset of cows on Days 0, 7, 10, and 11 to determine ovulations, presence of corpus luteum, and follicle diameter and in all cows 32 d after TAI for pregnancy determination. In 35 cows, plasma progesterone concentrations were determined 0, 3, 4, 5, 6, 7, and 12 d after ovulation. The proportion of noncyclic cows and cows with ovarian cysts on Day 0 were 12% and 6%, respectively. Ovulatory response to first treatment was 62% versus 44% for pLH and GnRH and 78% versus 50% for noncyclic and cyclic cows (P<0.01). Location, ovulatory response to first pLH or GnRH, cyclic status, presence of an ovarian cyst, and preovulatory follicle size did not affect pregnancy rate. Plasma progesterone concentrations after TAI did not differ among treatments. Pregnancy rate to TAI was greater (P<0.01) in the GnRH/PGF/pLH group (42%) than in the other three groups (28%, 30%, and 26% for GnRH/PGF/GnRH, pLH/PGF/GnRH, and pLH/PGF/pLH, respectively). Although only 3% of cows given pLH in lieu of GnRH on Day 9 lost their embryo versus 7% in those subjected to a conventional TAI using two GnRH treatments, the difference was not statistically significant. In summary, pLH treatment on Day 0 increased ovulatory response but not pregnancy rate. Cows treated with GnRH/PGF/pLH had the highest pregnancy rate to TAI, but progesterone concentrations after TAI were not increased. In addition, preovulatory follicle diameter did not affect pregnancy rate.

Gonadotropin releasing hormone receptor gene and protein expression and immunohistochemical localization in bovine uterus and oviducts.

Recently GnRH, GnRH-R systems has been demonstrated in various extrahypothalamic and extrapituitary reproductive tissues in different mammalian species, where GnRH acts in an autocrine and or paracrine manner and modulates different biological processes. GnRH-R mRNA has also been demonstrated in bovine ovaries (follicle and corpus luteum) and normal and carcinogenic human endometrium/endometrial cells. This is the first study elucidating presence of GnRH-R mRNA and GnRH-R protein in bovine uterus and oviducts in follicular and luteal phases of the estrous cycle and further localizing the receptors to endometrial and oviductal epithelial cells. To our knowledge this is the first report demonstrating GnRH-R mRNA and protein in mammalian oviducts. We used gene-specific primers and monoclonal GnRH-R antibody to test GnRH-R mRNA and GnRH-R protein through RT-PCR and immunobloting. Immunohistochemistry was employed to localize these receptors to endometrial and oviductal epithelial cells. GnRH-R mRNA and receptor protein were expressed at expected molecular weights of 920bp and 60kD, respectively. Densitometry analysis revealed that expression levels for GnRH-R protein in uterus and oviducts were similar to bovine pituitary. The presence of GnRH receptors in bovine uterus and oviducts is intriguing and it would be imperative to examine the functional role of this system in the regulation of reproductive processes.

Ovarian follicular and corpus luteum changes, progesterone concentrations, estrus and ovulation following estradiol benzoate/progesterone based treatment protocol in cross-bred cows.

The objectives of the present study were to investigate the effects of the stage of the estrous cycle at the start of an estradiol benzoate (EB) and progesterone (P) based treatment protocol on new follicular wave emergence, subsequent estrus and ovulation. The experiment was conducted using a crossover design with each cow (five cross-bred cows) being assigned to one of three groups at 3-month intervals within a 1-year period. Estrous cycle stage in individual cows was initially synchronized with prostaglandin F(2)alpha. After detection of estrus, each cow was injected intramuscularly (i.m.) with 2 mg EB and 200 mg P (EB/P) on day 5, 12 or 17 of the estrous cycle (estrus=day 0), followed by 1 mg EB i.m. 12 days after the EB/P treatment. Ovarian ultrasonographic examinations showed that the emergence of a new follicular wave occurred after EB/P treatment in all groups and the mean interval from EB/P treatment to wave emergence did not differ among the groups (3.2-3.8 days). All cows in each group exhibited behavioral estrus and ovulated the newly formed dominant follicle. However, cows in the day-17 group exhibited estrus 1-3 days before the second EB injection. The concentrations of progesterone showed faster reduction, during the treatment period, in the day-12 and -17 groups compared to the day-5 group. These results indicate that the EB/P treatment induces an emergence of a new follicular wave, irrespective of the estrous cycle stage at the start of treatment, but the effect of EB/P protocol on estrous/ovulation synchronization is influenced by the stage of the estrous cycle.

Progesterone (CIDR)-based timed AI protocols using GnRH, porcine LH or estradiol cypionate for dairy heifers: ovarian and endocrine responses and pregnancy rates.

The overall objective was to compare the efficacy of GnRH, porcine LH (pLH) and estradiol cypionate (ECP), in a modified Ovsynch/fixed-time AI (FTAI) protocol that included a controlled internal drug [progesterone] release (CIDR) device. In Experiment 1, heifers received a CIDR on Day -10, and PGF (25mg) on Day -3. At CIDR insertion, heifers received 100 microg of GnRH (n=6), 0.5mg of ECP (n=6), 5.0mg of pLH (n=6) or 2 mL of saline (n=7); these treatments were repeated on Day -1, except for ECP, that was repeated on Day -2, concurrent with CIDR-removal. The 5.0 mg pLH was the least effective with a longer interval to ovulation than the other groups combined (102 versus 64 h; P<0.05). Overall mean LH concentrations (1.6 ng/mL) and area under the curve (AUC) did not differ among treatments, but mean peak LH concentration was lower in heifers given 5 mg of pLH compared to all other groups (4.5 versus 10.3 ng/mL; P<0.05). In Experiment 2, heifers on CIDR-based Ovsynch protocols were given 12.5mg pLH (n=6; pLH-low), 25.0 mg pLH (n=6, pLH-high), or 100 microg GnRH (n=5; control). Heifers in the pLH-high group had greater (P<0.01) plasma LH concentrations (between 12 and 20 h) than GnRH-treated heifers, but the pLH treatments did not differ (P>0.10). Area under the curve for LH (ng/32 h) was at least 50% greater (P<0.01) in pLH-treated heifers compared to GnRH-treated heifers (mean, 41.3, 56.3 and 20.3 for pLH-low, pLH-high and GnRH, respectively). Ovulation occurred in 15 of 17 heifers. Progesterone concentrations were higher on Days 9 and 14 in heifers given 25mg of pLH, suggesting enhanced CL function. In Experiment 3, 240 heifers were assigned to CIDR-based Ovsynch/FTAI protocols. The first and second hormonal treatments (with an intervening PGF treatment on Day -3) were GnRH/GnRH (100 microg), ECP/ECP (0.5 mg), pLH/pLH (12.5 mg) or GnRH/ECP, respectively; pregnancy rates were 58.7, 66.1, 45.9 and 48.3%, respectively (ECP/ECP>both pLH/pLH and GnRH/ECP; P

GnRH in non-hypothalamic reproductive tissues.

Gonadotropin releasing hormone (GnRH) is a hypothalamic neuronal secretory decapeptide that plays a pivotal role in mammalian reproduction. GnRH and its analogues are used extensively in the treatment of hormone dependent diseases and assisted reproductive technology. Fourteen structural variants and three different forms of GnRH, named as hypothalamic GnRH or GnRH-I, mid brain GnRH or GnRH-II and GnRH-III across various species of protochordates and vertebrates have been recognised. The hormone acts by binding to cell surface transmembrane G protein coupled receptors (GPCRs) and activates Gq/11 subfamily of G proteins. Although hypothalamus and pituitary are the principal source and target sites for GnRH, several reports have recently suggested extra-hypothalamic GnRH and GnRH receptors in various reproductive tissues such as ovaries, placenta, endometrium, oviducts, testes, prostrate, and mammary glands. GnRH-II appears to be predominantly expressed in extra pituitary reproductive tissues where it produces its effect by PLC, PKA2, PLD, and AC cell signalling pathways. In these tissues, GnRH is considered to act by autocrine or paracrine manner and regulate ovarian steroidogenesis by having stimulatory as well as inhibitory effect on the production of steroid hormones and apoptosis in ovarian follicle and corpus luteum. In male gonads, GnRH has been shown to cause a direct stimulatory effect on basal steroidogenesis and an inhibitory effect on gonadotropin-stimulated androgen biosynthesis. Recent studies have shown that GnRH is more abundantly present in ovarian, endometrial and prostrate carcinomas. The presence of type-II GnRH receptors in reproductive tissues (e.g. gonads, prostrate, endometrium, oviduct, placenta, and mammary glands) suggests existence of distinct role(s) for type-II GnRH molecule in these tissues. The existence of different GnRH forms indicates the presence of distinctive cognate receptors types in vertebrates and is a productive area of research and may contribute to the development of new generation of GnRH analogues with highly selective and controlled action on different reproductive tissues and the target-specific GnRH analogues could be developed.

Growth factors and growth hormone enhance in vitro embryo production and post-thaw survival of vitrified bovine blastocysts.

The objective of this study was to assess the influence of specific growth factors and growth hormone (GH) in the culture medium on in vitro embryo production and post-thaw survival of vitrified blastocysts. In total, 1673 bovine oocytes were used for evaluating the nuclear status of the oocytes after in vitro maturation (n=560) or for in vitro fertilization (IVF, n=1113) and distributed in five treatment groups: (1). medium only control; (2). activin (10 ng/ml); (3). epidermal growth factor (EGF) (10 ng/ml); (4). insulin 5 microg/ml and (5). GH (100 ng/ml). There was an increase (P<0.05 and P<0.01, respectively) in the percentage of oocytes that reached meta phase II, developed to blastocysts and hatched, as well as in the blastocyst cell number in the groups treated with activin, EGF and GH compared to controls. There was no significant difference between insulin and control groups. A total of 465 blastocysts were vitrified in a three-step protocol using ethylene glycol and polyvinylpyrrolidone. After thawing, embryos were cultured in five treatments groups as described above. Groups EGF and GH had higher (P<0.05) survival rates with a mean blastocyst survival of 95.0+/-1.5 and 93.1+/-3.5%, respectively, while mean hatching rate was higher for EGF and activin groups (75.3+/-3.4 and 62.0+/-3.2%, respectively). Thawed control blastocysts had a mean cell count of 52.7+/-3.3%. With the exception of insulin, all growth factors and GH tested showed higher (P<0.01) total cell numbers when compared to controls. In conclusion, addition of growth factors and GH in the culture media has favorable effects on in vitro maturation, in vitro embryo production, and post-thaw survival of vitrified blastocysts.

Involvement of apoptosis in the atresia of nonovulatory dominant follicle during the bovine estrous cycle.

The present study was designed to 1) investigate whether apoptosis is responsible for the atresia of nonovulatory dominant follicle (DF), 2) to determine if atresia of a nonovulatory DF is associated with alterations in Bcl-2 and Bax expression, 3) to test whether progesterone P(4) has a direct effect on apoptosis in bovine follicles, and 4) to study the pattern of expression of Bcl-2 and Bax in follicles at different developmental stages (small, medium, and large). In experiment 1, 16 cycling cows received a norgestomet ear implant at proestrus (Day 1) for 9 days to mimic the subluteal phase. The cows were assigned either to a control (n = 4) or P(4)-treated groups (n = 12). Injections of P(4) (150 mg, i.m.) were given on Day 3 (n = 4); on Days 3 and 4 (n = 4), and on Days 3, 4, and 5 (n = 4) of the implant period. Controls received injections of corn oil on Days 3, 4, and 5. Unilateral ovariectomy was performed on Days 4, 5, and 6 to recover DFs from cows that had been treated with P(4) for 24, 48, and 72 h, respectively. DFs in the control group were collected on Day 6. The onset of atresia of DFs was assessed morphologically by ultrasound to determine DF diameters, histologically by light microscopic inspection of tissue sections, and functionally by quantification of follicular fluid steroid hormone levels. Apoptosis was detected by DNA analysis and in situ TUNEL labeling. Expression of Bcl-2 and Bax proteins was examined by Western blot analysis. The earliest signs of atresia were detected 24 h after P(4) injection as evidenced by decreased diameter, degeneration and detachment of granulosa cells (GCs) from the basal lamina, and a dramatically reduced ratio of estrogen to P(4). Electrophoretic analysis of DNA extracted from DFs of cows treated with P(4) for 24 h revealed a distinct ladder pattern of DNA fragments. In contrast, this pattern was not obvious in DFs from control cows. Similar results were also obtained from TUNEL analysis of DFs. Furthermore, both Bcl-2 and Bax were found to be present in all DFs; however, the ratio of Bcl-2 and Bax protein levels was significantly reduced by 24 h of P(4) treatment compared with DFs from the control group (P < 0.05). Experiment 2 investigated the direct effect of P(4) (4 ng/ml) on apoptosis of cultured GCs using ovaries obtained from a local slaughterhouse. In addition, the pattern of expressions of Bcl-2 and Bax in follicles at different developmental stages (small, medium, and large) was studied. No increase in apoptotic DNA fragments was detected in GCs treated with P(4). The ratio of Bcl-2 and Bax protein levels was variable in small follicles; however, Bax protein level was always relatively higher than that of Bcl-2 in medium and large follicles. In conclusion, our study suggests that apoptosis is the mechanism that underlies the atresia of nonovulatory DFs that develops during the luteal phase of bovine estrous cycle.

Morphological and biochemical identification of apoptosis in small, medium, and large bovine follicles and the effects of follicle-stimulating hormone and insulin-like growth factor-I on spontaneous apoptosis in cultured bovine granulosa cells.

The first objective of this study was to determine whether the death of bovine granulosa cells (GC) isolated from small ( 8 mm) follicles during follicular atresia occurs by apoptosis. The second objective was to establish an in vitro model system to elucidate the developmental (GC from follicles of different sizes) and hormonal (FSH and insulin-like growth factor-I [IGF-I]) regulation of bovine GC apoptosis during follicular atresia. Bovine ovaries were obtained from a nearby slaughterhouse. Follicles were classified by morphometric criteria as healthy or atretic. Apoptosis in GC from follicles of different sizes was analyzed by both morphological and biochemical methods. Bovine GC were cultured for 48 h at a density of 5 x 10(6) cells/ml in serum-free media at 39 degrees C to determine the effects of FSH and IGF-I on apoptosis. The results showed that apoptosis occurred in GC from all sizes of follicles. Apoptosis in GC was also detected in some healthy follicles. Degenerate GC displayed the morphological characteristics of apoptosis, including nuclei with marginated chromatin, a single condensed nucleus, multiple nuclear fragments, and/or membrane-bound structures containing variable amounts of chromatin and/or cytoplasm (apoptotic bodies). All GC classified as apoptotic on the basis of their morphology contained fragmented DNA measured by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) technique. Cells that had undergone apoptosis were observed mainly in GC and in scattered theca cells. Throughout the GC layer, apoptotic cell death was more prevalent among antral GC than among mural GC. Interestingly, morphological results showed that no apoptosis occurred in cumulus cells. A time-dependent, spontaneous onset of apoptosis occurred in GC from small, medium, and large follicles during in vitro serum-free culture. The rate of DNA fragmentation in the culture of GC from small follicles was higher than that from medium and large follicles. FSH attenuated apoptotic cell death in GC from medium follicles more effectively than in those from small follicles. IGF-I also suppressed apoptosis in cultured GC from small follicles. In conclusion, this study showed that 1) GC death during bovine follicular development and atresia occurs by apoptosis; 2) apoptosis occurs in GC and theca cells; however, apoptosis does not occur in cumulus cells even in atretic antral follicles; 3) GC from all small, medium, and large follicles undergo spontaneous onset of apoptosis when cultured under serum-free conditions; and 4) FSH and IGF-I can attenuate apoptosis in cultured bovine GC.

Factors affecting reproductive performance in captive Mallard ducks.

Propagation of wild-strain Mallards (Anas platyrhynchos) in captivity is hindered by low egg fertility. Therefore, we studied the effects of captivity, age, mate choice and isolation on reproductive parameters of Mallards. Captive drakes had smaller immature testes than free-flying Mallard drakes. Captive yearling ducks weighed less than adult ducks at the beginning of the breeding season, but no differences were found between their initial clutch size, egg volume or number of clutches laid. Yearling pairs had lower egg fertility (7%) than adult pairs (80%). Egg fertility was higher (51 vs 21%) in self-chosen pairs than in randomly assigned pairs. Isolation of ducks, however did not influence egg production.

Plasma testosterone profiles, semen characteristics, and artificial insemination in yearling and adult captive Mallard ducks (Anas platyrhynchos).

Testosterone profiles and semen characteristics were determined using yearling and adult captive wild-strain Mallard (Anas platyrhynchos) drakes. Wild-strain Mallard hens were artificially inseminated by modifying a technique developed for domesticated poultry. In both adult and yearling drakes, there was a change in the concentration of circulating plasma testosterone during the reproductive season. Testosterone concentrations increased from basal levels in March, peaked in April, and decreased to basal levels in May. The decrease in testosterone concentration to basal level was 2 wk earlier in yearlings than in adults (P < 0.05). The decrease in testosterone concentration was associated with the onset of postnuptial molt. Semen volume (0.04 to 0.08 mL) and semen concentration (approximately 1.32 x 10(9) spermatozoa per milliliter) were not different between adult and yearling drakes (P > 0.05). Overall mean fertility for yearling and adult drakes obtained with artificial insemination was 70.4%. These results suggest that artificial insemination may be used successfully in the propagation of captive wild-strain Mallard ducks.

Effects of unilateral ovariectomy on follicular development and ovulation in cattle.

In a study of 4 cyclic dry cows (Trial I) and 6 cyclic puberal heifers (Trial II), unilateral ovariectomy increased the number of ovulatory follicles, did not alter the hormone profile, cycle length or the number of follicular waves. Ovarian follicular development in all 4 cows was monitored daily using transrectal ultrasonography until the day of ovulation, during which period daily blood samples were also taken from the tail vein for determination of plasma FSH, LH and P4 concentrations. Unilateral ovariectomy was performed on the day after ovulation and ovarian activity was again monitored daily (ultrasonography and blood sampling for FSH, LH and P4) for 2 consecutive cycles (8 cycles in all). Estrus in all 6 heifers was synchronized using 2 injections of PGF2 alpha given 12 d apart. Similarly, ovarian activity in the 6 puberal heifers was monitored daily using ultrasonography and blood sampling for 1 complete control cycle. Following estrus and ovulation the left ovary was removed in all the animals, and thereafter 1 complete cycle was followed. Mean cycle length, FSH, LH and P4 concentrations before and after unilateral ovariectomy were compared using paired sample t-test. The results show that unilateral ovariectomy neither altered the cycle length nor the number of follicular waves in the cows, but it increased the number of ovulatory follicles (2 follicles developed and ovulated in 6 of the 8 cycles). The mean diameter of the largest follicle was 16.1 +/- 0.9 mm and the second largest 12.5 +/- 0.9 mm. No significant (P > 0.05) differences were observed in FSH (0.72 +/- 0.09 vs 0.71 +/- 0.07), LH (0.42 +/- 0.1 vs 0.37 +/- 0.07) and P4 (2.8 +/- 0.6 vs 2.6 +/- 0.4) levels before and after unilateral ovariectomy. Of the 6 heifers, 5 had 2 waves and 1 heifer had 3 waves of follicular growth during the control cycle, and this pattern did not change after the procedure. Mean cycle length (20.7 +/- 0.9 vs 21 +/- 0.9) did not differ before and after unilateral ovariectomy, and 4 of the 6 heifers ovulated twin follicles following ovariectomy. The mean diameter of the largest follicle was 14.5 +/- 0.7 mm and second largest measured 12.1 +/- 0.8 mm. No significant (P > 0.05) differences were observed in FSH (0.16 +/- 0.09 vs 0.21 +/- 0.07), LH (0.11 +/- 0.1 vs 0.15 +/- 0.07) and P4 levels (3.6 +/- 0.26 vs 3.8 +/- 0.29) before and after unilateral ovariectomy. Based on these results, we conclude that unilateral ovariectomy is an ideal method for obtaining twin ovulations in cows and heifers.

Effects of buserelin injection and deslorelin (GnRH-agonist) implants on plasma progesterone, LH, accessory CL formation, follicle and corpus luteum dynamics in Holstein cows.

The influence of Buserelin injection and Deslorelin (a GnRH analogue) implants administered on Day 5 of the estrous cycle on plasma concentrations of LH and progesterone (P4), accessory CL formation, and follicle and CL dynamics was examined in nonlactating Holstein cows. On Day 5 (Day 1 = ovulation) following a synchronized estrus, 24 cows were assigned randomly (n = 4 per group) to receive 2 mL saline, i.m. (control), 8 micrograms, i.m. Buserelin or a subcutaneous Deslorelin (DES) implant in concentrations of 75 micrograms, 150 micrograms, 700 micrograms or 2100 micrograms. Blood samples were collected (for LH assay) at 30-min intervals for 2 h before and 12 h after GnRH-treatment from cows assigned to Buserelin, DES-700 micrograms and DES-2100 micrograms treatments and thereafter at 4-h intervals for 48 h. Beginning 24 h after treatment, ovaries were examined by ultrasound at 2-h intervals until ovulation was confirmed. Thereafter, ultrasonography and blood sampling (for P4 assay) was performed daily until a spontaneous ovulation before Day 45. A greater release of LH occurred in response to Deslorelin implants than to Buserelin injection (P < 0.01). Basal levels of LH between 12 and 48 h were higher in DES-700 micrograms group than in DES-2100 micrograms and Buserelin (P < 0.05). The first wave dominant follicle ovulated in all cows following GnRH treatment. Days to CL regression did not differ between treatments, but return to estrus was delayed (44.2 vs 27.2 d; P < 0.01) in cows of DES-2100 micrograms group. All GnRH treatments elevated plasma P4 concentrations, and the highest P4 responses were observed in the DES-700 micrograms and DES-2100 micrograms groups. The second follicular wave emerged earlier in GnRH-treated than in control cows (9.9 vs 12.8 d; P < 0.01). However, emergence of the third dominant follicle was delayed in cows of DES-2100 micrograms treatment (37.0 d) compared with DES-700 micrograms (22.2 d), Buserelin (17.8 d) or control (19.0 d). In conclusion, Deslorelin implants of 700 micrograms increased plasma P4 and LH concentrations and slightly delayed the emergence of the third dominant follicle. On the contrary, Deslorelin implants of 2100 micrograms drastically altered the P4 profiles and follicle dynamics.

Progesterone-induced atresia of the proestrous dominant follicle in the bovine ovary: changes in diameter, insulin-like growth factor system, aromatase activity, steroid hormones, and apoptotic index.

The objective was to determine whether there were decreases in insulin-like growth factors (IGF)-I and -II and increases in low-molecular-mass IGF-binding proteins (IGFBPs) in association with an inhibition of aromatase activity (AA) and follicular fluid estradiol (E2) production during progesterone (P4)-induced dominant follicle atresia in cattle. Twelve cycling cows received a norgestomet ear implant at proestrus for 9 days and were assigned to control (n = 3) or P4-treated (n = 9) groups. Injections of P4 (150 mg, i.m.) were given on Days 3 and 4; Days 3, 4, and 5; or Days 3, 4, 5, and 6 of the implant period. Controls received injections of corn oil on Days 3, 4, 5, and 6. Ultrasonography of the dominant follicle and blood sampling were done daily. Unilateral ovariectomy was done one day after the last injection. The experiment was repeated with the remaining ovary (6 follicles/treatment group). Granulosa cells were cultured with radiolabeled testosterone to measure AA. Steroid hormones, IGF-I, and IGF-II were measured in follicular fluid by RIA. The follicular fluid IGFBP profile was quantified by Western ligand blotting. P4-treated cows showed a drastic reduction in AA in the dominant follicles, and follicular fluid E2 was several times lower than in controls. Moreover, in P4-treated groups, concentrations of follicular fluid IGF-I and IGF-II were lower than in controls. The quantity of low-molecular-mass follicular fluid IGFBPs increased in P4-treated groups. Accumulation of low-molecular-mass IGFBPs with a reduction in IGFs may be a mechanism of dominant follicle atresia during the bovine estrous cycle.

Ultrasound-guided follicle aspiration and IVF in dairy cows treated with FSH after removal of the dominant follicle at different stages of the estrous cycle [corrected].

Recently, transvaginal ultrasound-guided follicle aspiration technology has been found to be of great value for in vitro fertilization (IVF) programs, even though the oocyte recovery rate and cleavage rate of transferrable embryos were low. In this study, we investigated the effect of the removal of the dominant follicle at different stages of the estrous cycle on the ovarian response of donor cows. Four experiments (EXPs) were devised. In EXP 1, 3 cows received 20 mg FSH on Day 1, ovulation occurred on Day 0, and on Day 3 follicles were aspirated. In EXP 2, the dominant follicle of the first wave was removed on Day 6 from 3 cows which received 20 mg FSH on Day 7 and on Day 9 follicles were aspirated. In EXP 3, 2 pregnant cows received 20 mg FSH on 70 d of pregnancy and 48 hr later follicles were aspirated a total of 5 times at 5-day intervals. In EXP 4, after ovulation on Day 0, 9 cows received 20 mg FSH on Days 8 to 14 of the estrous cycle and 48 hr after the last injection, follicles were aspirated once. The respective mean +/- SD numbers of aspirated follicles and recovered oocytes were higher (p < 0.01) in EXP 1 (13.4 +/- 1.7 and 8.7 +/- 2.3), EXP 2 (12.1 +/- 1.4 and 7.7 +/- 1.7) and EXP 3 (10.7 +/- 2.1 and 7.0 +/- 2.2) than in EXP 4 (5.8 +/- 2.3 and 3.1 +/- 1.6). The oocyte recovery rates were higher (p < 0.05) in EXP 1, EXP 2 and EXP 3 than in EXP 4. Similarly, the respective numbers of viable oocytes and cleavage rates were higher in EXP 1, EXP 2 and EXP 3 (6.0 +/- 1.3, 5.0 +/- 1.1 and 4.6 +/- 1.5 viable oocytes (p < 0.01); 66, 73 and 65% cleavage rates (p < 0.05)) than in EXP 4 (2.4 +/- 1.1; 46%). The numbers of morulae and blastocysts were higher (p < 0.05) in EXP 1, EXP 2 and EXP 3 than in EXP 4. In conclusion 1) removal of the dominant follicles from lactating and pregnant cows enabled viable oocytes to be recovered constantly and repeatedly by aspiration at different reproductive stages, and that viable blastocysts can be produced after IVF. 2) The presence or absence of a dominant follicle significantly affects the ovarian responses to FSH treatment. 3) This ultrasound-guided procedure proved to be an effective, repeatable and safe method for viable oocyte recovery from valuable pregnant donors.

Anti-bull sperm monoclonal antibodies: I. Identification of major antigenic domains of bull sperm and manifestation of interspecies cross-reactivity.

The overall objective of this series of experiments is to generate immunological markers that may elucidate bull sperm surface changes in vitro. Here we report the initial experiments of the study, involving the production and characterization of monoclonal antibodies (mAbs) again bull sperm. BALB/c mice were immunized with phosphate-buffered saline (PBS)-washed whole bull sperm, and their spleen cells were fused with NS-1 myeloma cells in two separate cell fusion experiments, resulting in the generation of 15 mAbs. The mAbs were specific to antigens of either the posterior tail or the head regions of bull sperm and detected five major domains of antigen localization in the bull sperm (apical crescent, equatorial band, principal acrosomal, whole head, and posterior tail). Eleven of the 13 head-specific mAbs recognized intra-acrosomal antigens, whereas 2 mAbs recognized antigens that were localized in the plasma membrane. One mAb specific to the tail region was of the IgM class; the remaining 14 mAbs were of the IgG class. They were all sperm specific, with no cross-reactivity to bovine oocytes or to any of the 12 bovine somatic tissues tested. The mAbs were not species specific, however, because 11, 10, 2, and 1 of the 15 mAbs cross-reacted with sheep, pig, mouse, and human sperm, respectively. None of the mAbs cross-reacted with rooster sperm. The cognate antigens of the 11 tested mAbs were of testicular origin, but several of them showed enhanced binding to epididymal sperm. In western blot analysis, 3 of the 13 mAbs tested identified more than one protein band (40-200 kDa). Seven others recognized proteins of > or = 200 kDa, whereas three mAbs recognized no proteins.

Anti-bull sperm monoclonal antibodies: II. Binding changes during capacitation and influence on sperm-zona interactions in vitro.

Anti-bull sperm monoclonal antibodies (mAbs), generated against intra-acrosomal and surface antigens, were evaluated for their functional significance. In experiment I, the influence of mAbs on the bovine sperm-oocyte interaction in vitro was tested on a total of 493 oocytes in either three or four replicate trials. Although the number of sperm bound per zona increased significantly over untreated control samples (23.6 +/- 5.6 vs. 10.0 + 2.4, mean +/- standard error [SE]; P < 0.001) in the presence of one surface-reacting mAb, other mAbs had no effect. Experiment II was designed to determine if the mAbs would detect capacitation-related changes of bull sperm in vitro. Bull sperm were incubated in capacitation medium (supplemented Tyrode's medium [TALP] plus 10 micrograms/ml heparin) for up to 4.5 hours. At 0 and at 4 hours, mAbs in hybridoma culture supernatant were incubated for 30 minutes with sperm, labeled with fluorescein isothiocyanate (FITC)-conjugated secondary antibody, and processed for indirect immunofluorescence assay. Four mAbs specific to intra-acrosomal antigens exhibited a time-dependent increase (P < 0.05) in binding to bull sperm incubated under capacitation conditions. In contrast, the binding of the mAbs specific to surface antigens significantly decreased (P < 0.05) after 4 hours in the presence of heparin. Sperm viability did not change during the 4-hour period. In experiment III, mAbs specific to intra-acrosomal antigens were evaluated to assess bull sperm acrosomal status following lysophosphatidylcholine-induced acrosome reaction. A significant decrease (P < 0.01) in mAb binding following the induced acrosome reaction was observed with all the mAbs; it was highly correlated (r > or = 0.85; P < 0.01) with Pisum sativum agglutinin binding and Giemsa staining. The results suggest that some of the mAbs are potential biological markers for bull sperm surface changes associated with capacitation and the acrosome reaction in vitro.

Viability of bovine blastocysts obtained after 7, 8 or 9 days of culture in vitro following vitrification and one-step rehydration.

This study examined morphological appearance, viability and hatching rates in relation to the total cell number following vitrification of in vitro produced bovine blastocysts and expanded blastocysts. In Experiment 1, embryos obtained after 7, 8 or 9 d of culture were pooled and equilibrated in either 10% ethylene glycol (EG) or 10% EG plus 0.3M trehalose in Dulbecco's phosphate buffered saline (DPBS) supplemented with 10% calf serum and 0.6% BSA for 5 min each, at room temperature, and then vitrified together in precooled vitrification solutions consisting of 40% EG (Treatment 1), 40% EG plus 0.3M trehalose (Treatment 2), 40% EG plus 0.3M trehalose and 20% polyvinylpyrrolidone (PVP, Treatment 3) in DPBS. The embryo viability and hatching rates of Treatment 1 (19 and 3%) differed significantly (P < 0.05) from those of Treatment 2 (56 and 31%) and Treatment 3 (70 and 43%). There was a significant difference (P < 0.05) in embryo viability between Treatment 2 (31%) and Treatment 3 (43%). In Experiment 2, Day 7, 8 and 9 embryos were vitrified separately, with higher viability and hatching rates in Experiment 1 than in Experiment 2. The viabilities of Day 7 (87%), 8 (71%) and 9 (46%) embryos differed significantly (P < 0.05). Again, there were significant differences (P < 0.01) among the hatching rates of Day 7 (75%), 8 (38%) and 9 (9%) embryos. The total cell number of hatched blastocysts was then determined by differential fluorochrome staining. The total cell number of Day 7, 8 and 9 embryos differed significantly (P < 0.05).