Presence Of Corpus Luteum Research Articles

Comparison of long-term progestin-based protocols to synchronize estrus before fixed-time artificial insemination in beef heifers1

Two experiments were conducted to compare pregnancy rates resulting from fixed-time AI (FTAI) after administration of 1 of 2 long-term controlled internal drug release (CIDR)-based protocols. Heifers were assigned to treatment by age, BW, and pubertal status. The CIDR Select-treated heifers (Exp. 1, n = 37; Exp. 2, n = 192) received a CIDR (1.38 g of progesterone) from d 0 to 14, followed by 100 µg of GnRH, intramuscularly (i.m.) 9 d after CIDR removal (d 23) and PGF(2α) (25 mg, i.m.) 7 d after GnRH treatment (d 30). Heifers assigned to the Show-Me-Synch protocol (Exp. 1, n = 40; Exp. 2, n = 200) received a CIDR from d 0 to 14, followed by PGF(2α) 16 d later (d 30). Artificial insemination was performed at 72 or 66 h after PGF(2α) treatment for the CIDR Select- and Show-Me-Synch-treated heifers, respectively, and each heifer was given GnRH (100 µg, i.m.) at the time of AI. In Exp. 1, ovaries of each heifer were examined by transrectal ultrasonography on d 23 and 30 to characterize follicular dynamics. Follicles ≥5 mm and the presence of corpora lutea were recorded. On d 25, ovaries of each heifer were examined to characterize the status of dominant follicles recorded on d 23. Heifers were fitted with HeatWatch (DDx Inc., Denver, CO) estrus-detection transmitters at PGF(2α) to characterize estrus distribution up to FTAI. The diameter of dominant follicles on d 23 at PGF(2α) and on d 30, and the estrous response after PGF(2α) treatment up to the point of FTAI did not differ between CIDR Select- and Show-Me-Synch-treated heifers. Concentrations of progesterone in serum at PGF(2α) were greater (P = 0.07) in Show-Me-Synch- than CIDR Select-treated heifers (6.0 vs. 4.8 ng/mL, respectively). Pregnancy rates of heifers resulting from FTAI did not differ (P = 0.33) between CIDR Select- and Show-Me-Synch-treated heifers (CIDR Select, 59%; Show-Me-Synch, 70%). In Exp. 2, FTAI pregnancy rates tended (P = 0.07) to be greater in Show-Me-Synch-treated (62%) than in CIDR Select-treated (51%) heifers. Pregnancy rates at the end of the breeding season did not differ (P = 0.72; CIDR Select, 85%; Show-Me-Synch, 83%) between treatments. In summary, pregnancy rates resulting from FTAI were comparable for heifers assigned to each of the 2 long-term progestin-based protocols. The reduced treatment cost and animal handling associated with administration of the Show-Me-Synch protocol offer distinct advantages over the CIDR Select protocol despite similarities in pregnancy rates resulting from FTAI.

Inseminação artificial em tempo fixo e diagnóstico precoce de gestação em vacas leiteiras mestiças

Avaliou-se, durante um ano, o desempenho reprodutivo de 94 vacas leiteiras mestiças Bos taurus x Bos indicus submetidas a um programa de reprodução assistida. Um protocolo de inseminação artificial em tempo fixo (IATF) foi executado por meio de dispositivo intravaginal contendo progesterona e das injeções de prostaglandina F2α e de cipionato de estradiol. Por meio de ultrassonografia, entre 7 e 14 dias após as inseminações ou montas controladas, realizou-se a detecção de corpo lúteo nos ovários a fim de determinar a taxa de ovulação e, no 28º dia, fez-se o diagnóstico de gestação para cálculo da taxa de concepção. Respeitou-se um período mínimo de 34 dias após o parto antes do tratamento. Não houve influência do escore de condição corporal e da presença de corpo lúteo no início do protocolo, nem da reutilização do dispositivo intravaginal e da monta controlada ou inseminação artificial, sobre as taxas de ovulação, concepção e concepção das vacas ovuladas. As taxas de concepção e de concepção das vacas ovuladas foram afetadas negativamente pelo elevado número de dias pós-parto (DPP), ou dias em lactação e pela época quente do ano, primavera/verão. A resposta ao protocolo de inseminação artificial em tempo fixo baseado no uso de progesterona, PGF2α e cipionato de estradiol é prejudicada pelo aumento dos dias em lactação e pela época quente do ano. A condição corporal não afeta a resposta ao protocolo de inseminação artificial, desde que as vacas tratadas apresentem escore acima de 2,25 pontos.

Onset of puberty and early-life reproduction in Angus females divergently selected for post-weaning residual feed intake

Angus cattle that had been divergently selected for Low and High post-weaning residual feed intake (RFI) were used in two projects to evaluate early-life reproductive performance and onset of puberty in females. In the first project, data on the 1999-born females (n = 64) were evaluated for weight, subcutaneous fat (P8 fat depth) and reproductive performance over two breeding cycles. These females that were the result of 1.0–2.5 (mean of 1.8) generations of selection, had mean (±s.e.) post-weaning RFI of –0.82 ± 0.19 kg/day and 0.57 ± 0.18 kg/day for the Low and High RFI selection lines, respectively. In the second project, ultrasonography was used to scan the ovaries of the 2008-born heifers (n = 121) on four occasions following weaning. In these heifers the presence of corpus luteum provided evidence of ovulation, and hence the commencement of onset of puberty. The average of the estimated breeding value (EBV) for RFI of the parents of the Low RFI and the High RFI selection line heifers were –0.72 ± 0.05 and 0.37 ± 0.03 kg/day, respectively. In the first project, no significant selection line differences were evident for weight, age at first mating, pregnancy and calving rates (percentage of females that were pregnant, or the percentage that calved out of the total number used for mating), calf birthweight and weight of calf born per female exposed to bull. Females from the Low RFI line had significantly (P < 0.05) lower P8 fat depth relative to their High RFI contemporaries at most of the measurement dates (e.g. 9.2 ± 0.5 versus 12.0 ± 0.5 mm at the start of first mating). Low RFI females also calved significantly (P < 0.05) later in the calving season than High RFI females (35.7 ± 3.0 versus 27.6 ± 2.4 days). The results indicate that there is a delayed pregnancy date during the first mating season leading to a later calving date for the Low RFI heifers. The later first calving date was then maintained at subsequent calving. The later calving, however, did not impact on pregnancy and calving rates. In the second project, it was observed that irrespective of selection line, heifers that had attained onset of puberty had significantly (P < 0.05) greater P8 fat depth than those that had not. Hence the expectation was that, relative to High RFI heifers, the Low RFI heifers with their lower P8 fat, will attain onset of puberty at a slightly older age. This expected trend was observed but the difference was not significant, and further investigations are recommended.

Effect of body condition and suckling restriction with and without presence of the calf on cow and calf performance

Nutrition and suckling are largely recognised as the most important factors affecting the postpartum period and consequently the reproductive efficiency of beef cattle. The aim of this study was to evaluate the effects of body condition score (BCS) and suckling restriction with and without the presence of the calf on milk production, reproductive efficiency and calf performance. Sixty-three crossbred (Angus × Hereford) multiparous cows were managed to maintain different BCS at calving and thereafter (low vs moderate; L, n = 31 and M, n = 32). Within each group of BCS (L and M) at week 9 postpartum (66 ± 0.88 days postpartum) cows were assigned to three suckling treatments (ST): (i) suckling ad libitum (S, n = 20); (ii) calves fitted with nose plates during 14 days remaining with their dams (NP, n = 22); and (iii) calves were completely removed from their dams for 14 days, and thereafter returned (CR, n = 21). Milk production was assessed by milking procedure at Day 65 (the day before onset of ST) and every 20–22 days until the end of the experiment. Cows were bled via jugular venipuncture every 28 days from Day –98 (Day 0 = calving) until Day 66. From Day 66 cows were bled every 7 days until the end of the mating period (Day 128). Concentrations of progesterone, non-esterified fatty acids and β-hydroxybutyrate acid and insulin were measured. Presence of corpus luteum (CL) was recorded and maximum follicle diameter was measured in all cows from the onset of the ST (Day 66) and during the following 4 weeks (until Day 94) in a weekly frequency. At Day 94, more cows (P < 0.001) in NP and in CR had CL compared with S cows (68, 57 and 21% for NP, CR and S, respectively). At that time, more cows in M-BCS presented CL than cows in L-BCS (77 vs 25; P < 0.0001). Within M-BCS, there were no differences in milk production between ST groups, while L-BCS cows with NP or CR produced less milk than S cows. Calf liveweight at weaning was 159.3 ± 3.1, 150.1 ± 2.9 and 147.0 ± 3.1 kg for S, NP and CR, respectively (P < 0.001). Suckling restriction with and without the presence of the calf had similar effects on reproductive performance, milk production and calf growth, while BCS interacted with ST to influence milk production. These results indicate that temporary suckling restriction could be an excellent management tool to increase reproductive performance of cows in moderate condition.

106 PREGNANCY RATE AFTER EMBRYO TRANSFER OF IN VIVO-PRODUCED OVINE EMBRYOS CRYOPRESERVED BY SLOW FREEZING OR VITRIFICATION

Although slow freezing is the method of choice to cryopreserve in vivo-produced ovine embryos, vitrification has became an alternative procedure mostly developed for in vitro-produced bovine embryos. The aim of this work was to compare pregnancy rates after cryopreservation of in vivo-produced ovine embryos with slow freezing or open pulled straw (OPS) vitrification method. Ewes were synchronized using intravaginal sponges containing 60 mg of medroxyprogesterone acetate for 14 d. Superovulation was performed using a total dose of 176 IU of ovine FSH (Ovagen), in 6 decreasing doses (i.m.) from Day 12 to 14 of treatment (Day 0 = sponge placing). Ewes were hand mated with 2 rams of proven fertility. Embryos were recovered 6 days after estrous detection by surgical procedure, evaluated under stereomicroscope, and randomly assigned to the cryopreservation treatments. Slow freezing was performed in D-PBS supplemented with 1.78 M ethylene glycol, 0.1 M sucrose, 4 mg mL-1 of BSA, and 20% serum. Embryos were loaded into 0.25-mL plastic straws and placed into a -7°C methanol bath chamber. After seeding embryos were cooled to -35°C at a rate of 0.5°C/min and then stored in liquid nitrogen. Thawing was performed by placing the straws in a 30°C water bath for 30 sec. Vitrification was performed by using the OPS method (Vajta et al. 1998) with minor modifications. Embryos were incubated in D-PBS supplemented with 1.78 M ethylene glycol, 1.3 M DMSO for 3 min and then transferred for 25 s in vitrification solution of D-PBS with 3.56 M ethylene glycol, 2.6 M DMSO, and 0.5 M sucrose, loaded in a 1 mL drop in the OPS, and immediately submerged into and stored in liquid nitrogen. Warming was performed in D-PBS plus 0.25 M sucrose for 5 min and then into D-PBS plus 0.15 M sucrose for another 5 min. Before embryo transfer, the presence of corpus luteum (CL) was detected by laparoscopic examination. One embryo per recipient was surgically transferred in the apical extreme of the uterine horn ipsilateral to the CL. Pregnancies were determined by ultrasonography 41 days after embryo transfer. Data were analyzed using the chi-square test. We found 47.8% pregnancy rate using slow freezing (11/23) and 43.5% pregnancy rate using OPS vitrification (10/23). Statistical differences were not detected (P = 0.09). We conclude that vitrification by OPS system, with minor modifications, is a suitable procedure for in vivo-produced ovine embryo cryopreservation.

Non Cat‐Like Ovarian Cycle in the Eurasian and the Iberian Lynx – Ultrasonographical and Endocrinological Analysis

The Iberian lynx is considered the most endangered felid species. Therefore, an ex situ conservation program was initiated to protect this species from extinction. Additional knowledge on lynx reproduction biology and reliable methods for reproductive monitoring are important for developing a captive breeding program. The aim of this study in lynx was to implement transrectal ultrasonography to visualize ovarian structures (follicles, corpora lutea) and to assess ovarian activity in addition to analysis of serum progesterone and oestradiol. Because of limited access to Iberian lynxes, the less-endangered Eurasian lynx and bobcat were also studied in this comparative study. Recent endocrinological studies based on faecal and urinary progesterone and oestrogen metabolites revealed that steroid profiles in both these species were alike and did not follow the typical pattern of other felids. Pregnancy diagnosis was not possible, since progesterone concentrations did not differ between pregnant and pseudopregnant animals. Progesterone was also detected after parturition as well as after weaning until the onset of a new oestrous cycle. In the present study, the presence of corpora lutea during the non-breeding season was confirmed by ultrasonography and by elevated serum levels of progesterone averaging 3.56 +/- 1.3 ng/ml in Eurasian and 6.1 +/- 0.26 ng/ml in Iberian lynx, respectively. The ultrasonographical findings on the ovarian structures suggest strongly that corpora lutea developed after ovulation stay active until November and regress before the onset of the next oestrus.

Cisto ovariano em vacas de leite: incidência, resposta à aplicação de GnRH e desempenho reprodutivo

A incidência de cistos ovarianos, a resposta ao tratamento com GnRH e os efeitos da ocorrência de cisto no desempenho reprodutivo e na taxa de descarte foram determinados em vacas lactantes da raça Holandesa. Vacas lactantes (n=333) foram avaliadas semanalmente por ultrassonografia a partir da quarta semana pós-parto, visando à detecção de corpos lúteos (CL) e de folículos ovarianos maiores que 10mm. Na sétima semana pós-parto, as vacas foram classificadas: em ciclando (n=248; presença de CL em um dos exames ultrassonográficos); em anestro (n=54; ausência de CL e de folículos >25mm) e com cisto (n=31; ausência de CL e presença de estruturas >25mm), quando foram distribuídas em: grupo-controle (n=16; sem tratamento) e grupo-tratamento (n=15; vacas que receberam uma aplicação de GnRH). A taxa de cura foi de 60,0% no grupo das vacas tratadas e de 87,5% no grupo-controle. As vacas com cistos apresentaram maior intervalo parto-primeira inseminação artificial (P<0,05; 91,4±8,3 vs. 77,8±2,5), maior número de serviços por concepção (P<0,05; 4,4±1,2 vs. 3,6±1,5), maior intervalo parto-concepção (P<0,05; 214,8±25,9 vs. 174,9±7,7) e maior taxa de descarte (P<0,05; 41,2 vs. 21,8%) do que as vacas ciclando. Vacas diagnosticadas com cisto na sétima semana pós-parto apresentaram recuperação espontânea, embora o intervalo parto-concepção e a taxa de descarte tenham sido maiores para essas vacas.

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 2 × 2 factorial design, either 100 μg GnRH or 25 mg pLH im on Day 0, 500 μg cloprostenol (PGF) on Day 7, and GnRH or pLH on Day 9, with TAI 14 to 18 h 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.

154 REPRODUCTIVE SEASONALITY AND FOLLICULAR DYNAMICS IN THE PRZEWALSKI'S HORSE

Artificial insemination has served as a valuable tool for the genetic management of small, isolated populations of several wildlife species (Pukazhenthi B et al. 2004 Development 16, 33–46). The overall aim of the present project was to develop a successful protocol for AI in the Przewalski horse (Equus ferus przewalskii), which is formally listed on the IUCN Red List as “extinct in the wild.” Achieving the long-term goal requires a series of requisite studies to improve our fundamental understanding of reproductive biology in this rare species of equid; therefore, our specific objectives in the present project were to (1) characterize ejaculate traits and examine the influence of seasonality in stallions; and (2) determine the follicular dynamics in mares. Stallions (n = 7) were anesthetized and subjected to a standardized electroejaculation procedure (Collins CW et al. 2006 Anim. Reprod. Sci. 94, 46–49). Testicular volume (TTV) and seminal traits [total sperm concentration (TSC), and total (TM) and progressive sperm motility (PM)] were assessed once each season (winter, January to March; spring, April to June; summer, July to September; fall, October to December) for 1 year, with data analyzed by repeated-measures ANOVA. Mares (n = 8) were examined by ultrasonography in a specialized chute system 3 days/week during breeding season (April to June) over 3 years. At each examination, ovarian structures [size of the dominant follicle and presence of corpora lutea (CL)] were assessed. Values for total ejaculate volume (mean ± SEM, 33.0 ± 22.2 mL), sperm motility (TM, 58 ± 19%; PM, 49 ± 18%), and TSC (2.8 ± 0.3 × 109) were highly variable among individuals. Seasonality significantly (P &lt; 0.05) influenced TSC, which was highest in summer (4.41 ± 0.09 × 109) compared with spring (2.7 ± 0.5 × 109), fall (1.7 ± 0.4 × 109), and winter (2.5 ± 0.4 × 109). Season had no influence on TTV or other seminal traits (P &gt; 0.05), with motile sperm being recovered at all ejaculation attempts. Ultrasonography revealed that the reproductive cycle of the Przewalski mare was composed of two phases. The follicular phase (4.3 ± 2.2 days; range, 3 to 7) was characterized by the presence of one dominant follicle that grew at a rate of 2.9 ± 0.2 mm (range, 1–6 mm) per day and ovulated at a size of 43.8 ± 1.5 mm in diameter (range, 30–57 mm). The luteal phase was distinguished by the presence of a CL that was sustained and evident on the ovary for 12.2 ± 3.3 days (range, 9–15) before regression. In summary, the basic reproductive physiology of the Przewalski horse is comparable with the domestic horse, including the ability of stallions to retain year-round spermatogenesis and ovarian follicular dynamics (including number, size, and growth rate) in the mare. These data allow us to suggest that established protocols for AI in the domestic horse (including estrus induction) likely will have relevance to adapting this technology to the genetic management of the rare Przewalski horse. Morris Animal Foundation (Grant No. D07ZO-403), Sichel Endowment Fund, Smithsonian Endowment Fund, Animal Management staff at CRC and The Wilds.

Developmental Competence of Bovine Oocytes Selected by Brilliant Cresyl Blue Staining: Effect of the Presence of Corpus Luteum on Embryo Development

The local circumstances of the ovary may influenced by the presence of corpus luteum (CL). We investigated the cleavage rate and blastocyst development of bovine oocytes collected from ovaries with or without CL. Oocytes were incubated with brilliant cresyl blue (BCB) for 90 min and separated on the basis of low (BCB ) or high (BCB–) activity of glucose-6-phosphate dehydrogenase. For both types of ovaries, the embryo cleavage rates in the case of BCB oocytes were significantly higher than those in the case of BCB– oocytes, but were not higher than the rates in the control group. The percentage of blastocysts developing from BCB and BCB– oocytes from the ovaries with CL did not significantly differ. However, in the case of ovaries without CL, a significantly higher (P<0.05) percentage of blastocysts developed from BCB oocytes than from BCB– and control group oocytes. The presence or absence of CL did not significantly influence the cleavage rate and blastocyst development. Based on our results, we conclude that BCB staining facilitates the selection of competent oocytes that will develop into blastocysts for IVF better than conventional morphological selection methods. We also conclude that the CL does not significantly influence blastocyst development.

Expression of steroidogenic enzymes and synthesis of steroid hormones during development of ovarian follicles in prepubertal goats

Expression of mRNAs encoding cytochrome P450 side-chain cleavage (P450scc), cytochrome P450 17 α-hydroxylase (P450c17), and cytochrome P450 aromatase (P450arom) were characterized by the RT-PCR technique and concentrations of progesterone (P4), testosterone (T0) and estradiol (E2) were measured by radioimmunoassay during follicular development of prepubertal goats. Synthesis of mRNAs encoding P450scc and P450c17 began in preantral follicles, but mRNA encoding P450arom was not detectable until early antral formation. While mRNA for P450scc was expressed in both theca and granulosa cells, mRNA for P450c17 was expressed only in theca cells while P450arom mRNA only in granulosa cells. In nonatretic follicles from prepubertal ovaries, the relative quantity of mRNA expression of all the three enzymes increased with follicle size; however, while the concentration of P4 and E2 increased, that of T0 decreased with follicle size. While expression of mRNA encoding P450scc was unaffected, that of P450c17 mRNA decreased to the lowest level and mRNA for P450arom became undetectable following atresia; accordingly, while the concentration of P4 increased in the atretic medium follicles, that of T0 and E2 decreased to the lowest level after atresia. While the adult follicular stage follicles showed a similar cytochrome expression as the nonatretic follicles of prepubertal goats, the former contained higher levels of E2 and P4 than the latter. The presence of corpus luteum in an ovary decreased expression of P450scc, significantly in large follicles while it increased concentration of P4. These findings indicated that (1) similar to other species, changes in follicular steroid production in goats were explained in large measure by changes in steroidogenic enzyme expression; (2) while mRNA expression was similar, activities of some of the steroidogenic enzymes may differ between sexually mature and immature goats.

Reducing the Interval from Presynchronization to Initiation of Timed Artificial Insemination Improves Fertility in Dairy Cows

The objective was to determine if reducing the interval from presynchronization to the first GnRH injection (G1) of a timed artificial insemination (AI) protocol improves pregnancy per AI. One thousand two hundred fourteen Holstein cows, at 37±3 d in milk (DIM), were stratified by parity, DIM, and milk yield in the first month postpartum and randomly assigned to control (n=412), 2 injections of PGF2α at 37±3 and 51±3 DIM, then enrolled in a timed AI protocol 14 d later; PShort (n=410), 2 injections of PGF2α at 40±3 and 54±3 DIM, then enrolled in a timed AI protocol 11 d later; or PShortG (n=392), same as PShort, but with an injection of GnRH 7 d before G1. All cows received the same timed AI protocol (d 65, G1; d 72, PGF2α; d 73, 1mg of estradiol cypionate; d 75, AI). A subset of 1,000 cows had their ovaries examined by ultrasonography at G1 and 7 d later when PGF2α of the timed AI was given to determine presence of corpus luteum (CL) and ovulation to G1. Pregnancy was diagnosed on d 38 after timed AI, and pregnant cows were reevaluated for pregnancy 4 wk later. Altering the interval between presynchronization and G1 did not affect the proportion of cows with a CL at G1, but GnRH 7 d before G1 increased the proportion of cows with a CL. Ovulation to G1 was greater for 11 compared with the 14 d interval, but GnRH did not improve ovulation. The increased ovulation to G1 when the interval was reduced from 14 to 11 d was observed only in cows with a CL at G1, but treatment did not affect ovulation in cows without a CL at G1. Treatment affected the pregnancy per AI on d 38 and 66 after insemination, and they were greater for the 11 compared with 14-d interval, but addition of GnRH did not improve pregnancy per AI. Cows ovulating to G1 had greater pregnancy per AI regardless of whether or not they had a CL at G1. Reducing the interval from presynchronization to initiation of the timed AI protocol from 14 to 11 d increased ovulation to G1 and pregnancy per AI in lactating dairy cows.

Validation of a human progesterone enzyme immunoassay (EIA) kit for use on serum of cattle in Cameroon

A study aimed at validating a human progesterone enzyme immunoassay kit was carried out on cattle at Bambui, Cameroon. Progesterone ELISA Kits (EH-511) were obtained from Clinpro International. Forty-one cows were selected, of which 19 were pregnant and 22 within 14 days post partum. Blood samples were analysed and progesterone levels were deduced from a curve obtained from standard absorbance values (A450). Results show that 95.5% of postpartum cows had progesterone levels below 1 ng/ ml, with the highest level being 0.75 ng/ml. The mean level was 0.5 +/- 0.26 ng/ml. The cows in the 'pregnant group' had progesterone levels ranging from 3.5 to 17.5 ng/ml. This kit can be used for measuring progesterone levels in cattle. Levels of 1 ng/ml for two consecutive samples or one sample at or above 3 ng/ml are an indication of the presence of corpus luteum, while cows below 1 ng/ml will be in anoestrus.

Involvement of Plasma Progesterone, Oestradiol‐17β and Cortisol in Ovulatory Response to Gonadotropin‐releasing Hormone in Dairy Cows with Cystic Follicles

The aim of the present study was to examine the plasma concentrations of progesterone, oestradiol-17beta and cortisol in the cows with cystic follicle and to examine its relationship with the ovulatory response to gonadotropin-releasing hormone (GnRH). Eighty-five post-partum Holstein-Friesian cows with cystic follicles regardless of the presence of corpus luteum were studied. Follicular size, presence of corpus luteum and occurrence of ovulation were checked by palpation per rectum. Blood collection and palpation per rectum were conducted on days 0, 7 and 14. Gonadotropin-releasing hormone was administered at day 7. Plasma concentrations of progesterone, oestradiol-17beta and cortisol were determined. Progesterone concentrations of <3.2, 3.2-4.8 and >4.8 nmol/l were defined as low, intermediate and high, respectively. Sixty-three (74.1%) of 85 cows showed low (<3.2 nmol/l =1 ng/ml) progesterone concentrations on day 0. Only 40 (47.1%) of them showed low-low pattern of progesterone at days 0 and 7. In 27 (31.8%) of them, progesterone concentration had increased by day 14. Of 22 cows having high progesterone concentration (>/=4.8 nmol/l) on day 0, corpus luteum was not detected in 18 cows (21.2%). Only in 10 cows, cystic follicle disappeared after GnRH administration. However, only one of 27 cows in which progesterone pattern was low-low-high at days 0, 7 and 14 experienced ovulation of the cystic follicle. Significantly lower oestradiol-17beta concentration was found on day 7 in cows showing a low-low-low pattern than a low-low-high pattern of progesterone (43.0 +/- 4.6 vs 55.8 +/- 2.8 pmol/l, p < 0.05). There was no significant difference in cortisol concentration on any days (days 0, 7 and 14) between cows showing a low-low-low and low-low-high pattern of progesterone. These results suggest that approximately one-fifth of cows diagnosed to have ovarian cysts possess luteal cysts and that a high oestradiol-17beta concentration at the time of GnRH administration is involved in the subsequent ovulation of the follicle, although ovulated follicle may not be cystic.

Follicle Development during Luteal Phase and Altrenogest Treatment in Pigs

Synchronization of the oestrous cycle of gilts using altrenogest treatment has been found to increase ovulation rate. The current experiment investigated if the increase in ovulation rate after altrenogest treatment is related to increased follicle size at the end of altrenogest treatment compared with late luteal phase follicles. Crossbred gilts (n = 15) received altrenogest during 18 days [20 mg Regumate (Janssen Animal Health, Beerse, Belgium)], starting 5-7 days after onset of first oestrus. Control gilts (n = 15) did not receive altrenogest. At days 10-12 of the oestrous cycle [i.e. in the presence of corpora lutea (CL)], average follicle development was 2.51 +/- 0.20 mm (assessed with ultrasound) in altrenogest-treated gilts and 2.58 +/- 0.16 mm in control gilts (p > 0.10). During the last days of altrenogest treatment (i.e. when CL had gone into regression), average follicle size had increased to 3.01 +/- 0.31 mm (p < 0.05). Subsequent ovulation rate was 16.6 +/- 1.7 in altrenogest treated gilts and 15.1 +/- 1.2 in control gilts (p < 0.05). Altrenogest treatment resulted in increased follicle size after regression of the CL, showing that suppression of follicle growth by altrenogest alone is less severe than suppression by endogenous progesterone (either with or without altrenogest). Altrenogest treatment also resulted in a higher ovulation rate. However, it is unclear if the increased follicle size and higher ovulation rate after altrenogest treatment are causally related, as the relation between the two on an animal level was not significant.

132 SUCCESSFUL EMBRYO TRANSFER OF IN VIVO-PRODUCED RED DEER (CERVUS ELAPHUS) EMBRYOS AFTER CRYOPRESERVATION BY SLOW FREEZING OR VITRIFICATION

Multiple ovulation and embryo transfer (MOET) programs for red deer (Cervus elaphus) have been established commercially over the last decade, with embryo cryopreservation being a related practice necessary to enhance the use of valuable genetic information. The aim of this work was to establish alternative methods for red deer embryo cryopreservation by using slow freezing with ethylene glycol (SF–EG) and vitrification by open pulled straw (OPS) methods. After surgical flushing of 18 superstimulated donors, 54 transferable embryos were recovered; 28 were transferred fresh to synchronized recipients and the others were cryopreserved by SF–EG (n = 11) or OPS (n = 15), respectively thawed or warmed, and transferred to recipients. Fresh embryos were maintained in Dulbecco's PBS + 20% cow serum (holding medium, HM) until transfer (maximum 3 h after collection). SF–EG cryopreserved embryos were suspended in HM + 1.78 M EG + 0.1 M sucrose + 4 mg mL−1 BSA. After a 10-min equilibration, embryos were loaded individually into 0.25-mL plastic straws and placed into a −7°C methanol bath chamber. After seeding (5 min later), the straws were cooled from −7 to −35°C at a rate of 0.5°C min. Straws were plunged into and stored in liquid nitrogen. Thawing was performed by placing the straws in a 30°C water bath for 30 s; their contents were drained into HM until transfer. Embryos were vitrified using the OPS method with minor modifications. They were first incubated in HM + 1.78 M EG + 1.3 M DMSO for 3 min and then transferred for 25 s into a vitrification solution of HM + 3.56 M EG + 2.6 M DMSO + 0.5 M sucrose. Each embryo was loaded by touching a 1-µL drop with the straw, which was immediately submerged into and stored in liquid nitrogen. Warming was done by placing the narrow end of the straws into HM + 0.25 M sucrose for 5 min. Embryos were then transferred into HM + 0.15 M sucrose for 5 min and finally to HM until transfer. Both types of cryopreserved embryos were transferred a few hours after collection, immediately after thawing or warming. Before embryo transfer, the presence of corpus luteum (CL) of recipients was confirmed by laparoscopic examination. Each embryo was surgically transferred into the apical extreme of the uterine horn ipsilateral to the CL of one recipient. Pregnancy was determined by ultrasonography 41 days after embryo transfer. The pregnancy rate between groups was compared with the chi-square test (P &amp;lt; 0.05). No statistical differences were found between groups (Table 1). Our results show that both vitrification and slow freezing methods with EG are suitable to cryopreserve red deer embryos. Table 1. Pregnancy rates in recipient hinds after transfer of fresh, vitrified, or frozen red deer embryos

Recovery of large preantral follicles from buffalo ovary: Effect of season and corpus luteum

Preantral follicle can be considered as an alternative source of oocyte for in vitro production of embryos. The objective of the present study was to standardize a procedure for the isolation of large preantral follicles (>150–500 μm) from buffalo ovaries and to determine the effect of season and the presence of corpus luteum on the recovery rate of the large preantral follicles. A combined enzymatic cum mechanical approach was adopted to recover the large preantral follicles. In the first experiment, the ovarian cortical pieces were suspended in trypsin (1000–1500 BAEE units for milligrams of solid) and incubated at various temperatures for different periods, i.e. (1) trypsin (1%), 37 °C for 10 min; (2) trypsin (1%), 37 °C for 10 min + 4 °C for 3 h; (3) trypsin (0.5%), 37 °C for 20 min; (4) trypsin (0.25%), 37 °C for 20 min. Although there was no significant difference ( P > 0.05) among the different protocols, the first protocol yielded more follicles (3.2, 2.6, 1.8 and 1.5 per ovary, respectively). Hence, the first protocol was selected and used in the second and third experiments. In the second experiment, the effect of season, i.e. peak breeding season (October–March) versus low breeding season (April–September) was evaluated on the recovery rate of the large preantral follicles. The recovery rate of large preantral follicles from the ovaries during the peak breeding season was significantly ( P < 0.05) greater (9.92 ± 0.85 per ovary) than that of the low breeding season (4.95 ± 0.27 per ovary). In the third experiment, effect of the presence of corpus luteum on the recovery rate of large preantral follicles was studied. There was a significantly ( P < 0.05) higher yield of large preantral follicles from the ovaries with corpus luteum (8.05 ± 0.88 per ovary) than for the ovaries without corpus luteum (4.57 ± 0.43 per ovary). This study confirms that the large preantral follicles can be isolated from buffalo ovaries using a combination of enzymatic cum mechanical methods and that more large preantral follicles can be recovered during the peak breeding season and from the ovaries having corpus luteum.

Morphological Criteria of Bovine Ovaries for Predicting Retrieval Efficiency of Preantral Follicles

To predict the number of preantral (primordial, primary and secondary) follicles retrieved from bovine ovaries, we examined the relationship between morphological parameters of ovaries and number of preantral follicles retrieved mechanically. The preantral follicles were retrieved mechanically by slicing ovarian tissue and the influences of size of the ovaries, number of antral follicles, and presence of cystic follicle and corpus luteum on the retrieval were evaluated. Total 77 ovaries were used and significant (p5,637 vs. 3,689537) in the ovarian cortex. The retrieval number was significantly reduced in cystic ovaries compared with non-cystic ovaries (5,167825 vs. 20,6316,507). However, neither ovary size (4.5 cm) nor the presence of corpus luteum affected the follicle retrieval. In conclusion, the number of preantral follicles retrieved from the ovaries can simply be predicted by the number of antral follicles and the presence of cystic follicles in the ovarian cortex.

Reproductive seasonality in the Tete veld rat (Aethomys ineptus) (Rodentia: Muridae) from southern Africa

AbstractVery little is known about the reproductive biology of the recently recognized Tete veld rat Aethomys ineptus. In the present study, we investigated the seasonality of reproduction in this newly recognized rodent using a number of histological and endocrinological parameters. Body mass, reproductive tract morphometrics, gonadal histology, and plasma testosterone concentrations in males and plasma oestradiol‐17β and progesterone concentrations in females were assessed from a population in the north‐central part of South Africa over a 12‐month period in order to ascertain the pattern of reproduction in the species. Seminiferous tubule diameters in 59 males were significantly larger between September and February relative to between March and August. Although spermatogenesis was prevalent in the southern hemisphere winter (June–August), the number of spermatozoa in the epididymides decreased in the southern hemisphere spring (September–November), summer (December–February) and autumn (March–May). Testicular mass relative to body mass and testicular volume regressed between May and September but exhibited recrudescence between September and April, whereas plasma testosterone concentrations increased significantly between September and February relative to between March and August. Ovarian histology of 67 females showed corpora lutea to be present throughout the year, but decreased in number during winter, whereas mean plasma progesterone concentration increased significantly between August and November and again between February and April. This bimodal pattern of progesterone concentration suggests that up to two litters per breeding season may be raised by the Tete veld rat. Gravid females were found between October and April, whereas gravid or lactating females were conspicuously absent between May and September. Collation of all these data suggests that the Tete veld rat is a seasonal breeder with reproduction confined predominantly to summer and autumn months of the southern hemisphere. However, the presence of follicular development in females and the presence of corpora lutea outside the breeding season imply that the Tete veld rat may undergo spontaneous ovulation.

In vitro embryo production and embryo transfer in domestic and non-domestic cats

Over a 5-year interval, multiple laparoscopic oocyte retrievals were done in fishing cats ( Prionailurus viverrinus), caracals ( Caracal caracal) and domestic cats after ovarian stimulation with gonadotropins. From 21 retrievals in five fishing cats, 579 preovulatory oocytes (mean = 27.6) were recovered and 348 embryos were produced in vitro (mean = 16.6). A total of 452 preovulatory oocytes (mean = 25.1) were recovered from 18 of 24 retrievals in six caracals and 297 (mean = 16.6) embryos were produced. An additional 16 caracal embryos (19%) were produced after in vitro maturation of 83 oocytes, 59 of which came from six retrievals producing only immature oocytes. The presence of corpora lutea at oocyte retrieval occurred in each species (1) at a similar frequency (33%) and (2) more frequently during January through May (11 of 15 retrievals) than during the latter half of the year (4 of 30 retrievals). Of the 12 embryo transfer procedures done in fishing cats, one pregnancy (8%) was obtained and one live kitten born after the auto-transfer of 10 Day-6 embryos. In caracals, a total of 46 Day-4 or Day-5 embryos were auto-transferred to six recipients, one of which delivered two live kittens. Then, 109 caracal embryos were cryopreserved before thawing and transferring to nine recipients (mean = 12.1) on Days 5 or 6. From three pregnancies established (33%), a total of three kittens were born. Two to six gonadotropin treatments/oocyte retrievals were done in domestic cats during 1999 through 2003; an average of 24.9, 23.5, 22.0, 23.1, 23.5 and 40.9 oocytes ( P > 0.05) were recovered at the first through the sixth treatment cycles from 138, 138, 97, 49, 22, and seven retrievals, respectively.