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Biomedical subjects

R Rajamahendran

Publications and source records attributed to R Rajamahendran.

At least 19 recordsLinked to original sources
PubMed2006

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.

Animals↗
PubMed2005

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<or=0.05). In conclusion, CIDR-based Ovsynch/FTAI protocols using either GnRH/GnRH or ECP/ECP yielded pregnancy rates about 20% points higher than previously reported for dairy heifers bred to Ovsynch/FTAI in the absence of a CIDR.

Administration, Intravaginal↗
PubMed2005

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.

Animals↗
PubMed2003

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.

Activins↗
PubMed2000

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 (</= 4 mm), medium (5-8 mm), and large (> 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.

Animals↗
PubMed2000

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.

Animals↗
PubMed1999

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.

Aging↗
PubMed1998

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.

Animals↗
PubMed1998

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.

Animals↗
PubMed1998

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.

Aging↗
PubMed1997

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.

Animals↗
PubMed1997

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.

Animals↗
PubMed1996

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).

Journal Article↗
PubMed1996

Characteristics of corpus luteum formed from the first wave dominant follicle following hCG in cattle.

Two experiments were conducted to characterize the development and function of corpus luteum (CL) induced by hCG. In Experiment 1, cows (n = 18) were randomly assigned either to serve as controls (CONT, n = 6) or to receive hCG on Day 7 with (hCG-LUT, n = 6), or without (hCG-CONT, n = 6) surgical removal of the spontaneous CL on Day 12. The diameters of the hCG-induced and spontaneous CL of similar age did not differ (P > 0.05) between Days 1 and 4. At Day 5, the CONT (spontaneous) CL diameter (29.3 +/- 1.4 mm) was larger (P < 0.05) than that of the hCG-LUT (24.5 +/- 1.5 mm) or the hCG-CONT (24.6 +/- 1.7 mm) induced CL. Similarly, induced CL diameter for hCG-LUT and hCG-CONT groups was smaller (P < 0.01) than the CONT (spontaneous) CL between Days 10 to 14. Plasma progesterone (P(4)) levels were not different (P > 0.05) among treatment groups until Day 12. On Day 14, the P(4) concentration of hCG-LUT cows decreased (P < 0.01) to 1.1 +/- 0.9 ng/ml, then increased to 3.1 +/- 0.9 ng/ml by Day 18. Comparative values for hCG-CONT and CONT cows were 5.8 +/- 0.8 and 4.2 +/- 0.8; 4.5 +/- 0.8 and 5.5 +/- 0.8 ng/ml, respectively. The onset of regression of CL as well as estrous cycle length were similar (P > 0.05) for all treatment groups. In Experiment 2, the effects of intrauterine infusion of indomethacin on the diameter, function and life span of hCG-induced CL were examined. A slight, albeit not significant, suppression of PGFM levels was observed in indomethacin-infused cows (n = 4) compared with the controls (n = 4) in blood samples obtained once a day during the infusion period. However, in 2 cows from which blood samples were collected every 6 h, the control cow showed several pulses of PGFM while the indomethacin-treated cow exhibited none. Induced CL diameter and lifespan were not affected by indomethacin infusion. However, mean P(4) levels were higher (P < 0.05) between Days 16 and 20 in the indomethacin-infused group. In conclusion, the results suggest that 1) hCG-induced CL are functional but appear to be smaller and secrete less P(4) than spontaneous CL of similar age, and 2) the small size and reduced secretary function observed is not necessarily due to PGF(2alpha) secreted by the uterine endometrium but, probably, to inherent characteristics.

Journal Article↗
PubMed1996

Effect of hCG administration on day 7 of the estrous cycle on follicular dynamics and cycle length in cows.

The use of hCG in cattle at breeding or at different times after breeding has been associated with extension in estrous cycle length among cows that do not become pregnant. The objective of this study was to determine whether the increase in estrous cycle length observed in hCG-treated cows that fail to become pregnant is due to changes in ovarian follicular dynamics. Twelve nonbred lactating cows were randomly assigned either to receive hCG on Day 7 of the cycle (Day 0 = day of estrus, n = 6) or to serve as controls (n = 6). Ultrasound scanning was conducted daily from Day 0 until the onset of the next ovulation to monitor follicular and corpus luteum (CL) dynamics. Blood samples were collected for progesterone analysis at each ultrasound session. Ovulation of the Day 7 follicle occurred in all 6 hCG-treated cows. The time of emergence of the second-wave of follicular growth was advanced in hCG-treated cows but was not statistically different (P > 0.05) from that of the control cows (10.8 +/- 0.3 vs 12.7 +/- 1.4 d). The mean diameter of the second-wave dominant follicle from Days 15 to 18 was not different (P > 0.05) between the treatment groups. However, the second-wave dominant follicle had a slower growth rate (0.8 vs 1.3 mm/d) among cows treated with hCG compared with that of the controls. The second-wave dominant follicle was the ovulatory follicle in 5 control cows, but only in 3 hCG-treated cows. The dominant follicle from the third wave ovulated in 1 control and in 3 hCG-treated cows. The lifespan of the spontaneous CL and the time to low progesterone levels (< 1 ng/ml) were not different between the control and hCG-treated cows. These results suggest an altered follicular dynamic but no extension in estrous cycle length when hCG is administered on Day 7 of the cycle in postpartum cows.

Journal Article↗
PubMed1995

Binding of the anti-human sperm monoclonal antibody HS-11 to bull spermatozoa is correlated with fertility in vitro.

The present study aimed to determine if there is bull to bull variation in the binding of the anti-human sperm monoclonal antibody (MAb) HS-11 to bull spermatozoa, and to investigate if there is any correlation between HS-11 binding to spermatozoa and in vitro fertility of the bulls tested. Semen samples of a single collection (split frozen in 0.5-ml straws) from 8 dairy bulls were used. Swim-up separated motile spermatozoa were incubated in 90-microl drops of capacitation medium (TALP+10 microg/ml heparin) at 39 degrees C, 5% CO2, 95% air. At 0, 2, 4 and 6 h of incubation HS-11 was added (1:1000 final concentration), and the MAb binding was assessed by indirect immunofluorescence assay (IIFA). The HS-11 binding was indicated by a bright green fluorescence of the sperm acrosome region. In vitro-matured, good quality bovine oocytes were randomly allocated to spermatozoa of each bull for in vitro fertilization. Sperm samples of 2 to 3 bulls were used in each trial until 4 replicates per bull were attained for IVF (n approximately 100 oocytes/bull) and IIFA experiments. Sperm capacitation status was assessed simultaneously using an egg yolk lysophosphatidylcholine- (LC) induced acrosome reaction assay. The binding of HS-11 to spermatozoa was maximum at 4 h of incubation in most (6/8) of the bull semen samples. Significant (P < 0.01) differences were observed between bulls in the binding of HS-11 to their spermatozoa (range 22 +/- 8 to 52 +/- 5%) at 4 h, but not within replicates. Similarly, variations (P < 0.05) in the cleavage rate were also seen (range 22 +/- 9 to 58 +/- 7%) between bulls. The HS-11 binding and cleavage were significantly correlated (r = 0.43; n = 32; P < 0.05). The highest percentage of spermatozoa underwent acrosome reaction in response to LC treatment at the 4-h incubation period. This and the linear relationship between HS-11 binding and the cleavage rate observed in the present study together strengthen our earlier suggestion that the binding of the monoclonal antibody HS-11 to bull spermatozoa on a time-dependent manner, may indicate capacitation changes. We conclude that 1) between-bull differences exist in HS-11 binding to spermatozoa, and in the cleavage rate, and 2) HS-11 binding to spermatozoa is correlated with fertility, as determined by the cleavage of bovine oocytes matured and fertilized in vitro.

Journal Article↗
PubMed1994

Anti-human sperm monoclonal antibody HS-11: a potential marker to detect bovine sperm capacitation and acrosome reaction in vitro.

The crossreactivity between bull spermatozoa and monoclonal antibodies initially raised against mouse spermatozoa and human spermatozoa was tested by indirect immunofluorescent assay. The three anti-human spermatozoa monoclonal antibodies examined (HSK-9, HS-11, HS-63) crossreacted with methanol-fixed bull spermatozoa, whereas the anti-mouse spermatozoa monoclonal antibodies (MS-4 and MS-7) did not. A separate experiment was conducted to determine the binding ability of HSK-9, HS-11 and HS-63 with live (fresh) bull spermatozoa incubated (39 degrees C in CO2 incubator) in a capacitation medium (modified Tyrode's supplemented with 10 micrograms heparin ml-1). The binding of the monoclonal antibodies to the intra-acrosomal antigens of live bull spermatozoa was determined at 0, 2, 4, 6 and 8 h of incubation. At the beginning of incubation, binding was minimal (3.2 +/- 1.7%), but a much higher percentage of spermatozoa exhibited fluorescent staining after 2 h. The maximal binding was observed after incubation for 8 h (72.0 +/- 8.2%). The third experiment was performed to determine binding of HS-11 to frozen-thawed spermatozoa and to test whether there was any variation among bulls in HS-11 binding to spermatozoa, and to assess whether such binding is an indication of sperm capacitation. Frozen-thawed semen samples from five bulls were assessed for antibody binding after 0, 2, 4 and 6 h of incubation. Maximal binding was observed at 4 h. Lysophosphatidylcholine (100 micrograms ml-1) induced acrosome reaction assay was performed to assess sperm capacitation at various intervals.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrosome↗