Days Of FSH Treatment Research Articles

199 The effect of different doses of follicle-stimulating hormone during superovulation on ovarian function in dairy cattle

Cattle with an unknown antral follicle count (AFC) during follicular waves respond to different FSH doses during superovulation in a curvilinear fashion with the highest doses decreasing or not increasing ovulatory follicle number, number of transferable embryos, and number of corpora lutea (CL). These observations support the hypothesis that heifers treated with different FSH doses during superovulation will reach a superovulation maximum (SOVmax) and that doses exceeding SOVmax are excessive, resulting in a decrease in ovarian function. To test this hypothesis, we used eight 12-month-old Holstein heifers with a low AFC (<10 follicles ≥3mm in diameter, index for small ovarian reserve). These 8 heifers were subjected to 4 different superovulations with a 21-day intervals between each superovulation. During each superovulation, which began on Day 1 of a synchronized oestrous cycle before selection of a dominant follicle, 2 of the 8 heifers were treated (twice-daily FSH injections×4 days) with 20, 40, 80, or 120mg of Folltropin-V (Vetoquinol, Lure, France). At the end of the study, the same 8 heifers had been superovulated 4 times with each of the 4 FSH doses. To enhance ovulation rate in response to hCG, each superovulated heifer was injected with 3 different injections of prostaglandin F2α (PGF2α) spaced 12h apart to induce luteolysis of the original CL. Two PGF2α injections were given after each FSH injection on the last day of FSH treatment and the last PGF2α injection was given after the hCG (2500IU) injection, which was 12h after the last FSH injection. Daily ultrasonography was used to measure AFC and number of ovulatory follicles and CL during the study. Results of t-test analysis showed that at time of hCG, AFC was greater (P>0.05) for heifers treated with the 40-mg (mean±s.e.m., 27±3.7) v. 20-mg (17.9±2.5) FSH dose, but the response to higher FSH doses (27.9±4.4; 27±3.3) did not differ from the response to the 40-mg dose. Number of ovulatory follicles was greater (P<0.05) for heifers treated with the 40-mg (17.5±2.3) v. 20-mg (10.1±1.4) FSH dose, but the response to higher doses (18.9±3.3; 17.3±2.4) did not differ from the response to the 40-mg dose. At Day 7 post-hCG, CL number was greater (P<0.05) for heifers treated with the 40-mg (13.8±2.9) v. 20-mg (7.4±1.4) dose, but response to higher doses (9.9±3.2; 9.1±2.3) did not differ from the response to the 40-mg dose. Ovulation rate (ovulatory follicle number divided by CL number) was greater (P<0.05) for heifers treated with the 40-mg (79±0.08%) v. 80-mg (52±0.09%) or 120-mg (53±0.09%) dose, but similar to that of heifers treated with 20-mg (73±0.09%) FSH dose. These results support the conclusion that the 40-mg FSH dose achieves SOVmax and FSH doses greater than SOVmax in cattle with a low AFC and small ovarian reserve are excessive and detrimental to ovarian function. This project was supported by Agriculture and Food Research Initiative Competitive Grant no. 2017-67015-26084 from the USDA National Institute of Food and Agriculture (Washington, DC).

153 DETECTING PRE-OVULATORY LUTEINIZING HORMONE PEAKS IN ORDER TO OPTIMIZE THE RATIO OF VIABLE EMBRYOS USING PREDI′BOV®, A NEW ON-FARM OVULATION TEST

The LH peak in cattle is the most precise event for predicting ovulation beginning 24 h later, and thus, AI time. Previous studies demonstrated that embryo production was improved when AI was conducted 12 h before ovulation; that is, 12 h after LH peak. This study aimed to evaluate the benefit of LH peak monitoring with Predi′Bov® (ReproPharm®, Nouzilly, France) following superstimulation in order to optimize numbers of viable embryos (VE). Predi′Bov® is a rapid (40 min) and easy to use on-farm test allowing LH peak detection from a few drops of blood. The test was also used to estimate the variability in the time of the LH peak and onset of oestrus. This study was conducted by the embryo transfer teams of 3 French cooperatives, in collaboration with UNCEIA. Forty heifers in stations (Creavia, Midatest) and 23 cows on farms (GEN′Iatest) were superstimulated by 8 injections IM of Stimufol® or Pluset® (FSH1 to FSH8) over 4 days in 2011–2012. Donor station heifers were treated twice in a Latin square design with a reference protocol where AI was conducted 12 and 24 h after onset of oestrus, and in an experimental protocol where AI was conducted 12 and 24 h after a positive Predi′Bov® test. Semen of different sires was used for both protocols. The Predi′Bov® test was carried out on 3 blood samples (BS1,2,3) collected every 6 h beginning at FSH7 in stations and FSH8 on farms to detect the earliest LH peaks. To determine late LH peaks, Predi′Bov® test was carried out on BS4 collected 24 h after FSH8. Univariate statistical analysis was performed to look at the relationship between qualitative (chi-square) and quantitative (t-test) variables. The difference was considered significant when P < 0.05. The Predi′Bov® test showed that 37.5% (15/40) of LH peaks occurred during the last day of FSH treatment (BS1 or BS2) in stations and 26.1% (6/23) at BS1 on farms. At Creavia station (n = 24), the LH peak was detected anytime from 24 h before to 9 h after the onset of oestrus. In stations, the VE percentage did not differ whether AI was done following oestrus or LH peak detection (63.1% and 61.8% in reference and experimental protocols, respectively). In stations, the VE percentage from 9 females with an LH peak detected at FSH7 (BS1; 41%) in the reference protocol did not differ from the experimental protocol (50%). On farms, the VE percentage was numerically higher but not significant in the experimental protocol (65.4%, n = 16) compared to the reference protocol (47.2%, n = 7). Further investigations are needed, taking into account the effect of collection rank, sire, and female effects, to confirm the trends shown by these results. In conclusion, Predi′Bov® can be used as easily on farms as in stations. Its use allows the detection of animals that have early or late LH peaks, which in turn provides the opportunity of carrying out AI at the optimal time for such females.

224 EFFECT OF LENGTHENING THE SUPERSTIMULATORY TREATMENT PROTOCOL ON THE OVARIAN RESPONSE AND NUMBERS OF TRANSFERABLE EMBRYOS IN BEEF COWS

The present study was designed to test the hypothesis that an increase in the length of exposure of ovulatory follicles to progesterone and FSH during ovarian superstimulation will increase the number of ovulations and viable embryos in cattle. Twenty-four beef cows were initially subjected to follicle ablation at random stages of the oestrous cycle to determine the number of follicles at wave emergence; cows with comparable numbers of follicles were randomly allocated to groups by replicate. A single dose of prostaglandin F2α (PGF; Estroplan, Vétoquinol, QC, Canada) was given IM 7 to 9 days after follicle ablation and daily ultrasound examinations were performed to detect ovulation. Follicular ablation of all follicles ≥5 mm was done 5 to 8 days after ovulation and a progesterone-releasing intravaginal device (PRID, Vétoquinol) was inserted. The Control group (n = 12) was treated with 4 days of FSH and cows in the Long group (n = 12) were given 7 days of FSH treatment. The FSH treatments were initiated 1.5 days after ablation (Day 0) with a total dose of 400 mg of NIH-FSH-P1 (Folltropin-V; Bioniche Animal Health, Belleville ON, Canada). The total dose of FSH was distributed equally over 8 (Control) or 14 (Long) IM injections at 12-h intervals. Prostaglandin F2α was administered twice (at 12-h intervals) on Day 2 (Control) or Day 5 (Long) and PRID were removed 12 h after the last PGF. Both groups received 25 mg of porcine LH (pLH) IM (Lutropin-V; Bioniche Animal Health) 24 h after PRID removal and AI with frozen–thawed semen of proven fertility was done 12 and 24 h later. A third AI was done 12 h later in cows with 2 or more follicles ≥9 mm (12/12 and 9/12 in Control and Long groups, respectively; P = 0.22). All animals were subjected to transrectal ultrasonography every other day and at the time of ova or embryo collection. Ova or embryos were collected nonsurgically 7 days after the pLH injection and evaluated following IETS guidelines. Embryos were defined as transferable (Grades 1, 2 and 3) and freezable (Grades 1 and 2). Procedure Genmod was used to compare variables between treatments and results are presented as means ± SEM (Table 1). There was no significant difference in the total numbers of ova/embryos recovered, but there were more ovulations, corpora lutea (CL), fertilized ova and transferable and freezable embryos in the Long group (P < 0.05). Collection efficiency (number of ova/embryos over the number of CL) was lower in the Long group (P < 0.05). In summary, lengthening of the superstimulatory treatment resulted in an increased number of ovulations without a decrease in oocyte or embryo competence. Data suggest that the traditional 4-day superstimulatory treatment protocol provides inadequate time to maximize ovulatory response. Table 1.Superovulatory response (mean ± SEM) of cows subjected to a conventional 4-day (Control) or a 7-day (Long) treatment protocol

392 CHARACTERISTICS OF THE SUPEROVULATORY RESPONSE IN GYR (BOS INDICUS) CATTLE

Gyr is the most important zebu breed for dairy herds in Brazil and in other tropical countries. Superovulatory responses in this breed have been shown to be lower than in European or beef zebu breeds such as Nelore and Brahman. The aims of this study were to investigate the effect of the presence of a dominant follicle on the superovulatory response, and to determine endocrine patterns in superstimulated and non-superstimulated Gyr cows. The first experiment was designed to evaluate the effect of the dominant follicle on embryo yield. Multiparous, non-lactating Gyr cows were treated with a conventional superovulation (SOV) protocol [300 IU of Pluset (Serono, Roma, Italy) in 8 decreasing doses] starting on either Day 10 (G10, n = 14) or Day 8 (G8, n = 16) of the estrous cycle or 48 h after dominant follicle removal by ultrasound-guided follicle aspiration (G48, n = 10). Ovarian follicle populations were monitored daily by ultrasonography. Data were analyzed by ANOVA, and means were compared by Tukey's test. Dominant follicle removal resulted in a larger number of small follicles before SOV (27.1 ± 2.7 vs. 14.7 ± 1.5 and 13.1 ± 1.2; P < 0.05), but the number of follicles reaching a diameter larger than 9 mm after superstimulation (17.4 ± 1.3 vs. 14.4 ± 2.0 and 11.4 ± 2.0; P > 0.05), and the number of viable (IETS grades 1 and 2) embryos (3.1 ± 0.8 vs. 3.0 ± 0.7 and 3.3 ± 0.8; P > 0.05) did not differ from G10 and G8 groups, respectively. There was great variation in superovulatory response, and the Pearson correlation between follicle numbers at the time of initiating superstimulatory treatments and response was low (r = 0.49; P > 0.05). In the second experiment, endocrine patterns in superstimulated (n = 32) and non-superstimulated (n = 24) Gyr cows were compared. Blood samples were collected on Day 14 of the estrous cycle or after 4 days of FSH treatment when follicular fluid was also obtained from both groups by ultrasound-guided follicle aspiration. Plasma and follicular fluid samples were stored at −20°C until assay for progesterone (P4), androstenedione (A2), and estradiol (E2) by RIA, using commercial kits (MedLab, Auckland, New Zealand, and Diagnostic Systems Laboratories, Inc., Webster, MN, USA). Mean plasma E2 concentrations did not differ between FSH-treated and control cows (3.7 ± 0.4 vs. 3.2 ± 0.4 pg mL−1; P > 0.05). Intrafollicular concentrations of E2, A2, and P4 in FSH-stimulated follicles (mean size of 14.0 ± 1.2 mm) were 193.5 ± 83.0, 55.7 ± 17.0, and 54.8 ± 28.1 ng mL−1, respectively, lower (P < 0.05) than those found in non-superstimulated growing dominant (mean size of 11.6 ± 0.5 mm) follicles (501.2 ± 83.8, 122.2 ± 22.5, and 97.0 ± 21.9 ng mL−1, respectively), but similar to concentrations in non-superstimulated, non-dominant (mean size of 7.2 ± 0.4) follicles (152.6 ± 99.2, 37.7 ± 20.3, and 37.3 ± 6.8 ng mL−1 for E2, A2, and P4, respectively). Results suggest that factors other than follicle dominance adversely affect superovulatory responses in the Gyr breed, and that follicular steroidogenesis may also be adversely affected following treatment with exogenous FSH. Results also failed to support the use of ultrasonography to predict superovulatory response in the Gyr breed.

390 THE EFFECT OF REDUCED FSH DOSE AND NUMBER OF TREATMENTS ON SUPEROVULATORY RESPONSE IN CIDR-TREATED KOREAN NATIVE COWS

Reducing the total dose and numbers of treatments with FSH for superstimulation without decreasing embryo yield may be less stressful and more economical for bovine embryo transfer. The objective of this study was to investigate the effect of dose and the number of days of FSH treatment on superovulatory responses in CIDR-treated Korean native cows. Forty-two cows, at random stages of the estrous cycle, received a CIDR device (CIDRTM; InterAg, Hamilton, New Zealand), 1 mg estradiol benzoate (SY Esrone; Samyang, Seoul, Korea) and 50 mg progesterone (SY Ovaron; Samyang); gonadotropin treatment began 4 days later. Cows were divided into 2 groups based on the dose and numbers of days of treatment with porcine FSH (pFSH): T1 group (n = 20): a total of 28 mg pFSH (recommended dose of Antorin�; Kawasaki Pharmaceutical, Tokyo, Japan) was given in twice daily IM injections in decreasing doses over 4 days (5, 5, 4, 4, 3, 3, 2, and 2 mg); and T2 group (n = 22): a total of 24 mg pFSH given in twice daily decreasing doses over 3 days (5, 5, 4, 4, 3, and 3 mg). Otherwise, all cows received the same treatments. Twenty-five and 15 mg dinoprost (PGF2α; Lutalyse; Pharmacia & Upjohn, Puurs, Belgium) were given with the 5th and 6th injections of pFSH, respectively. CIDR devices were withdrawn with the 6th pFSH injection, and the cows received 100 �g Gonadorelin (GnRH; Fertagyl; Intervet, Boxmeer, The Netherlands) 36 h after CIDR device removal. Cows were artificially inseminated using commercial semen from 4 Korean native bulls twice, at 48 and 60 h after CIDR device removal, and embryos were recovered 6 or 7 days after the 2nd insemination. The number of CL was counted on the day of embryo recovery by transrectal ultrasonography (Sonovet 600 with 5.0 MHz linear-array transducer; Medison Co., Ltd., Seoul, Korea). The recovered embryos were evaluated according to the IETS Manual for stage of development and quality. All data between groups were compared using Student's t-test. The numbers of CL (9.7 � 1.1 vs. 9.4 � 1.3), total ova/embryos (7.2 � 1.1 vs. 6.3 � 1.4), transferable embryos (4.4 � 1.0 vs. 3.6 � 0.9), degenerate embryos (0.9 � 0.3 vs. 1.3 � 0.4), and unfertilized ova (2.0 � 0.6 vs. 1.5 � 0.5) did not differ between groups (T1 vs. T2), respectively (P > 0.05). Data indicate that the reduced dose (24 vs. 28 mg) and numbers of treatments (6 vs. 8) of pFSH for superstimulation of Korean native cows does not affect the embryo yield.

335EFFECT OF PATERNAL LINE OF DONOR ON SUPEROVULATORY RESPONSE AND EMBRYO QUALITY IN JAPANESE BLACK HEIFERS

The objective of this study was to investigate the effect of paternal line of donor on superovulatory response and embryo quality in Japanese Black cattle. Japanese Black cattle have paternal lines that can be classified into the following, according to meat quality and the growth rate: ‘high marbling meat type’, ‘high growth rate type’ and ‘high marbling meat and high growth rate (intermediate type)’. We hypothesized that paternal line of donor may affect superovulatory response. One hundred and forty superstimulations were performed on 56 Japanese Black heifers, and data on superovulatory response and embryo collection were analyzed. Japanese Black donors used in this study were from the following 4 paternal lines: Kedaka (large-framed with high growth rate;; n=9, 30 times), Fujiyoshi (intermediate type;; n=22, 47 times), Shigekane (high marbling meat type;; n=13, 40 times), and Tajiri (high marbling meat type and small-frame with slow growth rate;; n=12, 23 times). Donors were synchronized using a CIDR-B (EAZI-BREED, InterAg, NZ) for 8 to 12 days, and 500μg PGF analog (Resipron-C; Teikoku Zouki, Japan or Clopromate-C; Sumitomo, Japan) administered at CIDR removal. Superstimulation was initiated on Day 10 (Day 0=day of estrus) of the synchronized cycle;; FSH (Antrin R10;; Denka, Japan) was administered twice daily for 4 days with decreasing doses for a total of 28 Armour units FSH. PGF analog was administered in the morning on the last day of FSH treatment. Donors were given 50 or 100μg GnRH analog (Sporunen, Denka, or Conceral, Takeda Schering prau, Japan) at the time of estrus detection, and were inseminated 12h after the onset of estrus. Embryos were recovered on Day 7 after AI. The numbers of CL and follicles were recorded with transrectal ultrasonography immediately after embryo recovery. Embryos were classified according to the IETS Manual. Data were analyzed by Kruskal-Wallis test combined with Scheffe’s multiple comparison test. There were no significant differences in CL numbers among paternal lines: Kedaka (9.5±4.2), Fujiyoshi (8.5±5.1), Shigekane (7.8±5.0) and Tajiri (8.6±4.1). Mean number of recovered ova/embryos per donor was significantly (P<0.05) higher in the Kedaka line (8.4±5.1) than in the Fujiyoshi (4.9±4.1) and Shigekane (5.5±7.0) lines. The percentages of viable embryos was greater (P<0.05) in the Fujiyoshi (73.7%) and Shigekane lines (62.8%) than in the Tajiri line (50.9%), whereas the percentage of unfertilized oocytes was significantly (P<0.05) higher in the Tajiri line (41.5%) than in the Fujiyoshi (17.5%) or Shigekane (26.1%) lines. The percentages of freezeable and transferable embryos were not significantly different among paternal lines. Data suggest that the paternal line had an effect on the superstimulatory response in Japanese Black heifers, and especially the number and percentage of viable embryos per donor.

108CRYOPRESERVATION OF GERMPLASM FROM HERITAGE BREEDS OF DOMESTIC LIVESTOCK: SEASONAL EFFECTS ON SUPEROVULATION RESPONSE AND EMBRYO PRODUCTION

There is an urgent need to preserve the genetic diversity of rare breeds of domestic livestock. However, it is unknown if conventional superovulation methods are effective in unimproved breeds. The establishment of germplasm cryostorage banks requires the ability to produce viable preimplantation stage embryos. To our knowledge, we describe here the first successful production and cryopreservation of embryos from Gulf Coast Native (GCN) sheep and Tennessee Myotonic (TM) goats. Mature GCN ewes and TM does were synchronized (prostaglandin F2α, 7.5–10mg i.m.) and all TM does, and some GCN ewes, were treated with progesterone (20mg sq, 5×on alternate days, prior to FSH treatment, then 10mg i.m. on second day of FSH treatment). Animals were superovulated (FSH, 50–40–30mg bid i.m., decreasing over 3 days) both in April (cycling) and June (anestrous). Females were bred by natural service with a proven male over 24hr, and embryos were collected surgically from the uterus on Day 7. FSH-stimulated cycling TM does produced a total of 15.3±3.0CL and 10.7±7.2 morulae/animal, whereas FSH-treated anestrous TM does yielded 9.0±2.8CL and 7.0±8.4 embryos per animal. Recovered goat embryos were morphologically of good-excellent quality, and were cryopreserved using a conventional multi-step freezing protocol. Cycling GCN ewes responded to FSH simulation and produced 9.7±2.5CL per animal, whereas the ovaries of non-cycling ewes did not respond to exogenous FSH treatment (0CL). This report documents that TM does will effectively respond to FSH superovulation treatment and produce viable embryos during the breeding season and during the anestrous period. In contrast, GCN ewes appear to respond only to exogenous FSH during the breeding season. Collectively, these results demonstrate that the TM and GCN landrace breeds of goats and sheep display differential responses to hormone manipulation; yet, with breed-optimized protocols, preimplantation stage embryos can be obtained for long-term cryostorage of germplasm of heritage breeds of livestock. Supported by and in partnership with SVF Foundation.

Serum inhibin B levels measured early during FSH administration for IVF may be of value in predicting the number of oocytes to be retrieved in normal and low responders.

In a previous study we have found that in normal ovulatory women, serum inhibin B levels on days 4-6 of FSH administration correlated with the number of oocytes retrieved. In the current study we examined the significance of earlier inhibin B measurements in predicting the oocyte number, in both normal and low responders. Study A consisted of 19 patients undergoing their first IVF cycle (n = 10) or had a normal response ( vertical line 6 oocytes retrieved, n = 9), while study B consisted of 15 patients with a previous low ovarian response (<or=5 oocytes retrieved). All patients had day 3 FSH levels <10 IU/l. After pituitary suppression, 300 (study A) or 600 IU (study B) of pure FSH was administered daily. Serum FSH, inhibin A, inhibin B and estradiol (E(2)) were determined prior to and every 1-2 days throughout FSH treatment. Study A: oocyte number between 4 and 14 correlated significantly with serum inhibin B levels on all days of FSH treatment, and with inhibin A and E(2) late during treatment. No correlation was found between inhibin B and when oocyte number was >16. Study B: oocyte number correlated significantly with inhibin B and inhibin A on all days of FSH treatment, even on day 2 (r = 0.90, P < 0.001 and r = 0.65, P < 0.05 for inhibin B and A respectively). No significant correlation was found with E(2) levels. In both studies, all patients with inhibin B >100 pg/ml on treatment day 2 had >6 oocytes. Our data suggest that serum inhibin B measured early during FSH stimulation may indicate whether sufficient oocytes will be retrieved, in both normal and low responders. Serum inhibin B measured during early FSH treatment may be of predictive value in monitoring ovarian stimulation treatment for IVF.

Retinol administration to superovulated ewes improves in vitro embryonic viability.

Retinol and its metabolites, all-trans retinoic acid and 9-cis retinoid acid, are regulators of cellular growth, differentiation, and development and have been implicated in reproductive processes including folliculogenesis and embryonic survival. Three experiments were conducted to identify effects of retinoid treatment of superovulated ewes upon subsequent in vitro embryonic development. Ewes were treated with all-trans retinol (ROH), all-trans retinoic acid (RA), 9-cis retinoic acid (CIS), or vehicle (Control) on the first and last day of FSH treatment. Embryos were recovered at the morula stage, cultured in vitro for 96 h, and observed for blastocyst formation. Embryos from ROH-treated animals had a higher (p < 0.01) incidence of blastocyst formation than RA-, CIS-, or vehicle-treated animals (72% vs. 27%, 33% and 32%, respectively). In experiment 2, ewes were given ROH or vehicle and treated as above. ROH treatment resulted in an increased percentage of embryos forming blastocysts (70% vs. 22%, p < 0.05). In experiment 3, ewes were treated with ROH or vehicle, and embryos were collected at the 1- to 4-cell stage and cultured for 7 days. ROH treatment resulted in increased blastocyst formation (79% vs. 5%, p < 0.05). The majority of embryos (60% vs. 6%; p < 0.01)) from vehicle-treated animals failed to develop beyond the 8-cell stage in comparison with those from ROH animals. ROH treatment of superovulated ewes increased embryonic viability and positively impacted embryonic development.

Influence of varied progestogen treatments on ovarian follicle status and subsequent ovarian superstimulatory responses in cows.

The influence of ovarian follicle status and follicle dominance on the response to superstimulatory treatment with FSH was examined in cows. In Experiment 1, oestrus was synchronised using Crestar and on Days 4-6 of the ensuing oestrous cycle cows were assigned to: Group NO (n = 9), control, endogenous CL and no treatment; Group N1 (n = 15), injected with a luteolytic dose of cloprostenol (500 micrograms) and implanted with one implant (3 mg) of the synthetic progestogen, norgestomet; Group N8 (n = 18), injected with 500 micrograms cloprostenol and implanted with eight (24 mg) implants of norgestomet. On Days 9-11, seven implants were removed from six cows in Group N8 and these cows, plus eight Group N1 and all Group N0 cows, were superstimulated with porcine FSH (Folltropin-V) over 4 days (360 mg total dose). The remaining implants were removed from cows in Groups N1 and N8 on Days 11-13, and all cows received 500 micrograms cloprostenol. Numbers and sizes of ovarian follicles, and CL, were recorded by trans-rectal ultrasonography; the largest follicle > 10 mm in diameter was considered morphologically dominant (DF). On Days 9-11, the proportions of cows with a DF were: Group N0, 3/9; Group N1, 14/15; Group N8, 0/18. Total follicles on the 4th day of FSH treatment were greater (P < 0.05) for cows in Group N1 (21.6 +/- 4.2) compared with Group N0 (10.9 +/- 2.4), with cows in Group N8 (13.2 +/- 0.9) not different from the other two groups. Subsequent numbers of CL were lower (P < 0.05) for cows in Group N1 (5.0 +/- 1.3) compared with Group N0 (9.4 +/- 2.0), with cows in Group N8 (8.5 +/- 1.0) not different from the other two groups. In Experiment 2, oestrus was synchronised in cows and on Days 4-6, cows were assigned to: Group C0 (n = 7), control, endogenous CL and no treatment; Group C3 (n = 6), received three CIDR-B intra-vaginal devices that delivered progesterone. On Days 9-11, two CIDR-B were removed from cows in Group C3 and all cows treated with FSH as in Experiment 1. The remaining CIDR-B was removed from cows in Group C3 on Days 11-13 and all cows injected with 500 micrograms cloprostenol. Proportions of cows with a DF on Days 9-11 and diameter of largest follicle were: Group C0, 6/7 and 12.6 +/- 0.9 mm; Group C3, 2/6 and 9.6 +/- 0.8 mm. Numbers of CL on Day 8 after oestrus were: Group C0, 20.0 +/- 7.1; Group C3, 14.8 +/- 4.8 (P > 0.05). Exposure to low dose norgestomet allowed development of a persistent dominant follicle, resulting in a reduced response to superstimulation with FSH. High dose progestogen restricted follicle growth without apparent effects on responses to superstimulation.

Neonatal exposure of rats to recombinant follicle stimulating hormone increases adult Sertoli and spermatogenic cell numbers

It is believed that Sertoli cells support a finite number of germ cells and that Sertoli cell number may help determine the spermatogenic capacity of the adult. In the rat, Sertoli cells divide during fetal and early postnatal life, a process controlled in part by FSH. This study examined the effect of recombinant human FSH on postnatal testicular development and the impact of neonatal FSH exposure on the adult animal. Sprague-Dawley rats received FSH (200 IU/kg s.c.) daily from birth (Day 1) until Day 5, 10, 15, and 20. In a second experiment, animals received FSH for the first 10 or 15 days of life and were killed at Day 90. Sertoli and spermatogenic cell numbers were determined by stereological methods (the optical disector technique). FSH treatment significantly (p < 0.001) increased testicular weights (135%, 193%, and 173% of controls at Days 10, 15, and 20, respectively). The absolute volume of the epithelium and interstitium increased significantly as a result of increases in tubule diameter and length. In response to FSH treatment, the number of Sertoli cells increased significantly (p < 0.01) to 168%, 139%, and 151% of control numbers at Days 10, 15, and 20, respectively. After 15 days of FSH treatment, the numbers of spermatogonia and spermatocytes also increased (169% and 220% of control, p < 0.01, p < 0.001, respectively). The labeling index of Sertoli cell nuclei, as determined by bromodeoxyuridine incorporation, was unaffected by FSH treatment, with cessation of Sertoli cell division occurring as normal at Day 15. FSH treatment for the first 10 or 15 days of neonatal life resulted in testicular hypertrophy in adulthood (testis weight 118% and 124% of control, respectively). Similar increases were seen in the absolute volume of seminiferous epithelium and lumen, which were attributed to an increase in tubule length. Sertoli cell numbers increased to 113% and 149% of control after 10 and 15 days, respectively, of FSH exposure, with similar increases in round and elongated spermatid numbers. We conclude that exposure of the neonatal rat to recombinant FSH results in testicular hypertrophy with increases in seminiferous tubule volume and length and proportionate increases in Sertoli and germ cell numbers. This trophic effect of increased perinatal FSH exposure persists into adulthood to augment the spermatogenic potential of the rat.

The effects of recombinant follicle-stimulating hormone on the restoration of spermatogenesis in the gonadotropin-releasing hormone-immunized adult rat.

The role of FSH in spermatogenesis is unclear as testosterone alone has been reported to be sufficient in the gonadotropin-deficient rat. This study examined the effects of recombinant FSH on the restoration of spermatogenesis after gonadotropin withdrawal by GnRH immunization. Adult Sprague-Dawley rats received GnRH immunogen (100 micrograms, sc, every 4 weeks) to induce gonadotropin deficiency, with severe spermatogenic regression occurring by 12 weeks. Recombinant human FSH was then given (10 or 50 IU/kg, sc, daily) for 7, 14, and 21 days, with data from both dosages combined in the analyses. Testes were perfusion fixed, and germ cell numbers were quantified by the optical disector technique. After 7 days of FSH, testis weight significantly increased by 43% (P < 0.01), with no further increases at 14 and 21 days. GnRH immunization severely reduced germ cell numbers, which were then significantly (P < 0.05) restored in all cell types, except elongated spermatids, by 7 days of FSH; type A spermatogonia (45%-->61% of control), type B spermatogonia/preleptotene spermatocytes (46%-->65%), leptotene/zygotene spermatocytes (39%-->55%), pachytene spermatocytes in stages I-VIII (11%-->30% control) and IX-XIV (4.3%-->22% control), and round spermatids in stages I-VIII (1.4%-->4.4% control). Prolonged FSH treatment did not further increase type A spermatogonial or pachytene spermatocyte number, whereas round spermatids increased to a peak of 12.8% of the control value. At no stage did FSH increase elongated spermatid numbers above 1% of the control level. The incorporation of bromode-oxyuridine into spermatogonial and early spermatocyte nuclei did not change after GnRH immunization or FSH treatment. Sertoli cell number was not altered by any treatment; however, Sertoli cell nuclear volume was significantly decreased from the control value by GnRH immunization (142 +/- 9 vs. 455 +/- 22 microns 3; P < 0.01) and increased after 7 and 14 days of FSH treatment to 212 +/- 10 and 259 +/- 24 microns 3, respectively. FSH treatment restored serum inhibin levels to normal, but did not increase serum or testicular androgen levels. We conclude that recombinant FSH partially restores spermatogenesis in the gonadotropin-deficient rat by increasing the number of spermatogonia and promoting subsequent maturational steps up to the round spermatid stage. Spermatid elongation was not restored by FSH, indicating the need for an additional factor(s), most likely testosterone.

Origin of the follicular fluid added to the media during bovine IVM influences embryonic development

The objectives of this study were to evaluate the developmental competence of bovine oocytes matured in the presence of selected follicular fluids (bFF) harvested a) from large follicles of FSH-treated cows on the fourth day of FSH treatment; b) from dominant follicles during the growing or c) the regressing phase; and d) from a pool of small follicles (< 5 mm) or e) medium follicles (5 to 10 mm) from untreated ovaries. Selected oocytes obtained from ovaries collected at the slaughterhouse were washed and matured in TCM-199 supplemented with 10% of the above bFF and/or 10% bovine estrous serum (BES) and gonadotropins (LH and FSH). The oocytes were then fertilized in vitro and cultured with oviductal vesicles for 6 d. The resulting ova (n = 1793) were fixed and stained, and the number of nuclei was counted. The average number of nuclei per ovum at the > 16-cell (P < 0.07) and the > 32-cell stage (P < 0.05) was higher for those matured in the presence of bFF from dominant follicles than in bFF from superovulated animals, respectively. The overall proportion of ova reaching the > 16-, > 32-, > 64- or > 128-cell stage was similar for those matured in bFF and BES. The proportion of ova reaching the > 16-cell stage was higher (P < 0.04) for those matured in bFF from selected follicles (dominant or superovulated) than in bFF from pooled follicles. Among oocytes matured in the presence of pooled bFF, the proportion of those reaching the > 16- (P < 0.02) or the > 32-cell stage (P < 0.06) was lower for those matured in a pool of medium follicles than the pool of small follicles. The proportion of embryos reaching the 64-cell stage was higher (P < 0.04) for those matured in the presence of bFF from dominant follicles than in the presence of bFF from superovulated animals. Based on the evaluation of the number of nuclei per embryo, it is concluded that the origin of follicular fluid influences the ability of in vitro-matured oocytes to acquire developmental competence.

Restoration of Spermatogenesis by Follicle-Stimulating Hormone Despite Low Intratesticular Testosterone in Photoinhibited Hypogonadotropic Djungarian Hamsters (Phodopus Sungorus)1

By exposing male Djungarian hamsters to short-day photoperiods, a hypogonadotropic condition resembling that of hypophysectomized animals can be achieved without resorting to surgery. By use of this animal model, differential effects of gonadotropins on spermatogenesis have been studied revealing that FSH selectively restimulates spermatogenesis up to the level of spermatids. In the present study, we investigated whether FSH (6 IU/day) also restores fertility in hamsters exposed to short-day photoperiods (8L:16D). Animals kept under long-day photoperiod (16L:8D) served as controls. After 8 wk under 8L:16D, gonads were involuted and the animals (n = 9-10 per group) were treated with FSH, or testosterone implants, or both for the following 33 days. An additional group received FSH for 33 days and, for the last 12 days only, FSH plus testosterone to induce copulatory and ejaculatory behavior. The implants caused no increase in intratesticular testosterone as compared to that in short-day controls. All males were paired with adult females for the last 5 days of treatment, and killed thereafter. Litter sizes were determined 22 days later. The examination of the testes by flow-cytometrical techniques revealed qualitatively normal spermatogenesis in animals receiving FSH, whereas testosterone caused an insignificant increase to a few round spermatids. In most FSH-treated animals, sperm were present in the epididymides. In hamsters receiving FSH along with testosterone, treatment resulted in restoration of full fertility as compared to long-day controls. Fertility was also restored in animals receiving testosterone only for the last 12 days of FSH treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Spermatogenesis in larval Ambystoma tigrinum: Positive and negative interactions of FSH and testosterone

Hypophysectomized larval Ambystoma tigrinum were treated for 20 days with either saline, FSH, testosterone, or FSH+testosterone. Larvae from each of these groups were either killed on day 21 or treated for a second 20 days with either saline, FSH, testosterone, or FSH + testosterone. Testes from larvae treated with FSH for 40 days had large germinal cysts, many of which contained primary or secondary spermatocytes. The secondary spermatogonial cysts, which developed following 20 days of FSH-treatment, degenerated during the second 20-day period of experimentation unless FSH-treatment was continued. Depending on the testicular condition, testosterone inhibited or stimulated spermatogenesis. If testes were treated for at least 20 days with FSH + testosterone and did not receive prior testosterone-treatment, testosterone inhibited the FSH-induced proliferation of secondary spermatogonia. Conversely, if FSH+testosterone treatment followed treatment with testosterone, testosterone enhanced the FSH-induced spermatogenesis.

Formation of new corpora lutea in mature hypophysectomized rats.

New corpora lutea of oviolation were produced in mature hypophysectomized rats by a 4-day regimen of standard FSH and LH preparations. A wide range of gonadotrophin dosages caused luteinization of unruptured fol- licles, which resulted in the formation of corpora lutea atretica. However, oviolation and the forma- tion of 4 or more corpora lutea per rat could be induced consistently only with certain optimum dosages. The subsequent development and func- tion of the induced corpora lutea were also stud- ied. In the absence of further treatment, the in- duced luteal tissue in both types of luteal bodies developed into morphologically persistent but nonfunctional structures similar to those observed in untreated hypophysectomized rats. Prolactin administration, begun on the day after the in- duced ovulation, resulted in functional corpora lutea capable of supporting trauma-induced uterine deciduomata. (Endocrinology 78: 1259, 1966) T HE RELATIVE ease with which ovulation and corpus luteum formation may be in- duced by pituitary gonadotrophins in intact rats contrasts markedly with the results in hypophysectomized animals. This difference, noted in the first experiments with hypophysec- tomized rats (1), was confirmed and extended in later studies using crude preparations of pituitary gonadotrophins (2, 3). These later experiments demonstrated that both dosage and source of extract were critical factors in the induction of ovulation. On the other hand, luteinization of unruptured follicles, sometimes resulting in almost solid luteal bodies (corpora lutea atretica) containing trapped ova, was a consistent observation. Other experiments (4) with purified pituitary preparations revealed that normal ovulation and corpus luteum formation in hypophysecto- mized rats required 4 days of FSH treatment followed by at least 4 or 5 days of combined FSH and LH treatment. The investigators did not note any of the corpora lutea atretica ob- served previously with crude extracts. Carter et al. (5) also obtained normal ovulation and corpus luteum formation with purified prepara- tions. These authors used a treatment regimen approaching the physiological: 4 days of fol- licular development with an ovulatory stimulus on the fourth day. Furthermore, it was ob-