Stimulation of folliculogenesis in domestic cats with human FSH and LH

Folliculogenesis in response to exogenous stimulation by human urinary follicle stimulating hormone (huFSH) and human menopausal gonadotropin (hMG) was evaluated in the domestic queen (Felis catus). The role of LH and/or FSH in folliculogenesis was examined by measuring concentrations of estradiol 17beta (E(2)) and progesterone (P) in the serum. Additionally, changes in the number and size of follicles from before the administration of exogenous hormones to surgical oocyte collection were monitored. Findings indicated that in queens receiving huFSH or hMG followed by human chorionic gonadotropin (hCG) to induce ovulation, the numbers of follicles from 1 to 3 mm increase with statistical significance (P<0.005) from before the initiation of treatment to surgical collection of oocytes. Although E(2) concentrations in cats receiving hMG increased above baseline by the third exogenous hormone injection, mean E(2) concentrations did not increase in the groups that received both huFSH and hCG, or hCG only, until after the administration of hCG. This suggests that the exogenous administration of LH contained in both hMG and hCG was necessary for E(2) to rise to levels associated with estrus.     

Pregnancy in the domestic cat after vaginal or transcervical insemination with fresh and frozen semen

The objective was to compare pregnancy rates in domestic cats using fresh semen for intravaginal artificial insemination (IVI), either at the time of hCG treatment for induction of ovulation, or 28 h later, and to compare pregnancy rates following IVI or transcervical intrauterine insemination (IUI) of frozen-thawed semen. Eighteen queens were inseminated during 39 estrus cycles. Fresh semen with 13.5+/-5.4 x 10(6) sperm (range, 6.8-22 x 10(6)) collected by electroejaculation from four male cats was used in Experiment 1, and cryopreserved semen (20 x 10(6) sperm, with 70+/-5% post-thaw motility) from one male cat was used in Experiment 2. Serum concentrations of estradiol-17beta and progesterone were determined in most queens on the day of AI and again 30-40 days later. Treatment with 100 IU of hCG 3 days after the onset of estrus induced ovulation in 95% of treated queens. Pregnancy rates to IVI with fresh semen at the time of hCG administration versus 28 h later were not different (P=0.58); overall 33% (5/15) of the queens became pregnant. For frozen-thawed semen, AI was consistently done 28h after hCG administration; IUI and IVI resulted in pregnancy rates of 41.7% (5/12), whereas no queen (0/12) became pregnant by IVI (P=0.0083). In conclusion, an acceptable pregnancy rate was obtained with frozen-thawed semen in the domestic cat by non-surgical transcervical IUI; this method might also be useful in other small felids.     

GnRH agonist Lupron (leuprolide acetate) pre-treatments prevent ovulation in response to gonadotropin stimulation in the clouded leopard (Neofelis nebulosa)

In many species, controlling the ovary prior to induction of ovulation improves the success of ovarian response and artificial insemination (AI). We assessed the impact of suppression of estrus with the GnRH agonist, Lupron, on ovarian sensitivity to equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) in the clouded leopard. Seven female clouded leopards were given two injections of Lupron (3.75 mg IM) 23 d apart, followed 44 d later by eCG and hCG. Daily fecal samples were collected from 60 d before Lupron to 60 d after hCG. Fecal metabolites of estrogen (E) and progesterone (P) were measured by radioimmunoassay. Lupron decreased (P < 0.05) the number of E peaks during Lupron treatment compared to pre-Lupron. All females had baseline E and six of seven (86%) had nadir P on day of eCG. Exogenous gonadotropins induced E elevations in all females. However, mean E in the gonadotropin-provoked estrus was decreased (P < 0.05) compared to pre-Lupron estrous periods. Only one of seven (14%) females ovulated after eCG/hCG. In conclusion, estrous cycle control with Lupron resulted in predictable ovarian suppression prior to gonadotropin stimulation but altered ovarian sensitivity by an as yet unknown mechanism so that ovulation was inhibited, even when using a proven exogenous gonadotropin protocol.     

Ovarian activity reversibility after the use of deslorelin acetate as a short-term contraceptive in domestic queens

The objective was to evaluate ovarian activity reversibility in domestic queens after short-term contraceptive treatment with deslorelin acetate. Ten mature queens were used. In all queens, the estrous cycle was evaluated every 72 h by vaginal cytology (VC) and behavior assessments. When queens had VC characteristic of interestrus or diestrus, one deslorelin acetate implant (4.7 mg) was placed in the subcutaneous tissue of the interscapular region (day of insertion = Day 0). Thereafter, VC was performed every 48 h and on Day 90, implants were removed. At Day 100, estrus and ovulation were induced with 100 IU eCG (im), followed by 100 IU hCG (im), 84 h later (Day 103.5). Queens were ovariohysterectomized on Day 106. Corpora lutea (CL) were counted, oviducts were flushed, and oocytes were identified, isolated and stained to assess viability. In all queens, blood samples for plasma progesterone concentrations were collected once a week, from Days −21 to 106. After deslorelin acetate application, four queens had VC and behavior typical of estrus, and one ovulated. Furthermore, ovulation occurred in three queens that did not have VC or behavior consistent with estrus. After the initial ovarian stimulation, all females had anestrous VC during the deslorelin treatment period. Implants were readily removed. Following implant removal, all females responded to treatments to induce estrus and ovulation. There were (mean ± SEM) 13.1 ± 5.5 CL and 8.1 ± 5.5 oocytes per queen; the oocyte recovery rate was 56.8 ± 25.4% and all recovered oocytes were viable. We concluded that deslorelin acetate can be used as a reversible short-term contraceptive in domestic cats, because estrus and ovulation were successfully induced following implant removal.     

Ovarian response to human chorionic gonadotropin or gonadotropin releasing hormone in cats in natural or induced estrus

Human chorionic gonadotropin (hOG) or gonadotropin releasing hormone (GnRH) was given alone or with repeated coital stimuli to study ovarian activity and ovulation in the domestic cat. Adult cats in natural estrus (NE) or treated with follicle stimulating hormone (FSH-P) to induce estrus (2.0 mg/d for 5 d; IE) were assigned to one of five treatments: I, mating (M) only (three times daily for the first 3 d of estrus); II, M + hOG (250 IU, i.m. on Days 2 and 3 of estrus); III M + GnRH (25 mug, i.m. on Days 2 and 3 of estrus); IV, hOG only (250 IU, i.m. on Days 2 and 3 of estrus); or V, GnRH only (25 mug on Day 2 and 3 of estrus). Overall, IE females produced a greater (P < 0.05) number of corpora lutea (7.6 +/- 0.9) and unovulated follicles (18.9 +/- 2.1) than NE cats (4.9 +/- 0.6 and 3.6 +/- 0.9, respectively). For both NE and IE females, the M + hOG treatment (II) produced a greater number (P < 0.05) of ovulations (9.1 and 13.9, respectively) than any other ovulatory regimen (I, 4.1, 6.6; III, 4.1, 7.8; IV, 4.0, 6.2; V, 4.1, 5.6, respectively). These results indicate that 1) the excessive follicle number resulting from FSH-P treatment cannot be reduced with any of the hOG or GnRH treatments tested and 2) the use of hOG with copulatory stimuli synergistically enhances the ovulatory response of cats experiencing a natural estrus or those treated with FSH-P.     

Ovarian response in the estrual cat receiving varying dosages of HCG

Laparoscopy was utilized to determine the ovulatory response of the domestic cat to various dosages of human chorionic gonadotropin (HCG) administered intramuscularly at one or two time periods during estrus. A linear HCG dose-ovulatory response was observed in queens receiving 0--500 IU HCG as a single injection on day 1 or as injections given on days 1 and 2 of estrus. Animals treated with 500 IU HCG on day 1 or days 1 and 2 of estrus produced the maximum percent ovulation rates based on pre- and post-HCG ovarian morphology observation by laparoscopy (100.0, 95.9%, respectively). A single injection of 500 IU HCG produced a significant increase (p less than 0.05) in mean percent ovulation rate in comparison to the 0, 50 and 100 IU HCG single injections. Administration of 500 and 250 IU HCG for 2 days of estrus resulted in a greater percent ovulation rate than the 2-day injection of 50 IU (p less than 0.05). These results indicate that the proportion of mature follicles ovulating in an induced ovulator, such as the cat, is an increasing function of graded dosages of exogenous hormone.     

Estrus behavior and fecal steroid profiles in the Asiatic lion (Panthera leo persica) during natural and gonadotropin-induced estrus

In this paper the behavior of the Asiatic lion was studied during natural and gonadotropin-induced estrus in relation to fecal estradiol and progesterone concentration. The average length of estrus was 5.4 days and no significant difference was observed between natural and gonadotropin-induced estrus. Vocalization and rolling were the major estrus behavioral activities of Asiatic lions and the frequency of these activities were similar in both natural and gonadotropin-induced estrus and treatment with exogenous gonadotropin did not alter estrus behavioral activities. A significant positive correlation was observed between fecal estradiol and frequency of estrus behavior during natural and gonadotropin-induced estrus. Following gonadotropin treatment estrus could be induced in 69% of animals and these induced animals ovulated following hCG treatment. This study reports for the first time the successful use of the non-invasive fecal steroid assay for monitoring the induction of estrus and ovulation in the Asiatic lion.     

Synchronization of ovulation in cyclic gilts with porcine luteinizing hormone (pLH) and its effects on reproductive function

The overall objective was to evaluate the use of porcine luteinizing hormone (pLH) for synchronization of ovulation in cyclic gilts and its effect on reproductive function. In an initial study, four littermate pairs of cyclic gilts were given altrenogest (15 mg/d for 14 d). Gilts received 500 microg cloprostenol (Day 15), 600 IU equine chorionic gonadotropin (eCG) (Day 16) and either 5mg pLH or saline (Control) 80 h after eCG. Blood samples were collected every 4h, from 8h before pLH/saline treatment to the end of estrus. Following estrus detection, transcutaneous real-time ultrasonography and AI, all gilts were slaughtered 6d after the estimated time of ovulation. Peak plasma pLH concentrations (during the LH surge), as well as the amplitude of the LH surge, were greater in pLH-treated gilts than in the control (P=0.01). However, there were no significant differences between treatments in the timing and duration of estrus, or the timing of ovulation within the estrous period. In a second study, 45 cyclic gilts received altrenogest for 14-18d, 600 IU eCG (24h after last altrenogest), and 5mg pLH, 750 IU human chorionic gonadotropin (hCG), or saline, 80 h after eCG. For gilts given pLH or hCG, the diameter of the largest follicle before the onset of ovulation (mean+/-S.E.M.; 8.1+/-0.2 and 8.1+/-0.2mm, respectively) was smaller than in control gilts (8.6+/-0.2mm, P=0.05). The pLH and hCG groups ovulated sooner after treatment compared to the saline-treated group (43.2+/-2.5, 47.6+/-2.5 and 59.5+/-2.5h, respectively; P<0.01), with the most synchronous ovulation (P<0.01) in pLH-treated gilts. Embryo quality (total cell counts and embryo diameter) was not significantly different among groups. In conclusion, pLH reliably synchronized ovulation in cyclic gilts without significantly affecting embryo quality.     

Endocrine and ovulatory responses of the gilt to exogenous gonadotropins and estradiol during sexual maturation

Three studies were conducted to investigate the endocrine and ovulatory responses of the prepubertal gilt to exogenous estradiol and gonadotropins. In Study One, prepubertal gilts of 190 days of age were injected s.c. with pregnant mare's serum gonadotropin (PMSG) or physiological saline (SAL). Following PMSG injection, circulating levels of estradiol-17 beta (E2) increased. This increase was followed by a surge of luteinizing hormone (LH), estrus, a rise in progesterone (P4) levels, and ovulation. None of the gilts given SAL had increased levels of E2, LH or P4, and none ovulated. In Study Two, prepubertal gilts of 165 days of age were treated with varying doses of PMSG. A positive correlation was observed between dose of PMSG and peak levels of E2 (r = 0.83, P less than 0.001) and between dose of PMSG and number of corpora lutea (r = 0.96, P less than 0.001). In Study Three, gilts were treated at ages of 70 to 190 days with estradiol benzoate (EB), PMSG, or corn oil plus saline (CO/SAL) followed in 72 to 96 h by human chorionic gonadotropin (hCG) or SAL. All gilts treated with EB at 100 to 175 days of age had two surges of LH at an approximately 24-h interval. Gilts responding to EB at 70 and 190 days had only one surge of LH. Gilts of 100 days of age or older responded to PMSG with a single surge or two surges of LH. Ovulation in response to treatment was observed in gilts of 100 days of age or greater but not at 70 days. The conclusions drawn from these studies are that 1) PMSG-induced ovulation is preceded by an increase in circulating levels of E2 and in some gilts by a surge of LH, and 2) prepubertal gilts are able to respond to exogenous endocrine stimulation with either a single surge or multiple surges of LH at 70 to 190 days but are unable to ovulate in response to exogenous gonadotropins until 100 days of age.     

The effect of pre-ovulatory anaesthesia on ovulation in laparoscopically inseminated domestic cats.

Laparoscopic intrauterine artificial insemination (AI) of electroejaculated spermatozoa was used to compare embryo development and conception rates in domestic cats inseminated either before or after ovulation. Females were given a single (100 iu) injection of pregnant mares' serum gonadotrophin (PMSG) followed by either 75 or 100 iu human chorionic gonadotrophin (hCG) 80 h later. Cats were anaesthetized (injectable ketamine HCl/acepromazine plus gaseous halothane) 25-50 h after administration of hCG for laparoscopic assessment of ovarian activity and for transabdominal AI into the proximal aspect of the uterine lumen. At the time of AI, 23 cats were pre-ovulatory (25-33 h after hCG injection) and 30 were post-ovulatory (31-50 h after hCG injection). Pre-ovulatory females produced 10.5 +/- 1.1 follicles and no corpora lutea compared with 1.9 +/- 0.5 follicles and 7.5 +/- 0.9 corpora lutea for the post-ovulatory group (P < 0.05). Six days later, the ovaries of nine pre-ovulatory and 12 post-ovulatory females were re-examined and the reproductive tracts flushed. On this day, pre-ovulatory cats produced fewer corpora lutea (2.8 +/- 1.5; P < 0.05) and embryos (0.4 +/- 0.3; P < 0.05) than post-ovulatory females (18.9 +/- 3.3 corpora lutea; 4.6 +/- 1.2 embryos). Two of the 14 cats (14.3%) inseminated before ovulation and not flushed became pregnant compared with 9 of 18 cats (50.0%) inseminated after ovulation and up to 41 h after hCG injection (P < 0.05). These results indicate that ovulation in cats is compromised by pre-ovulatory ketamine HCl/acepromazine/halothane or laparoscopy or by both and that electroejaculated spermatozoa deposited by laparoscopy in utero, after ovulation, result in a relatively high incidence of pregnancy. Because ovulation usually occurs 25-27 h after injection of hCG, the lifespan for fertilization of the ovulated ovum appears to be at least 14 h in vivo in cats.     

Induction of estrus in pubertal miniature gilts

Genetic engineering of miniature pigs has facilitated the development of numerous biomedical applications, such as xenotransplantation and animal models for human diseases. Manipulation of the estrus is one of the essential techniques for the generation of transgenic offspring. The purpose of the present study was to establish a useful method for induction of the estrus in miniature gilts. A total of 38 pubertal miniature gilts derived from 4 different strains were treated with exogenous gonadotropins. Estrus and ovulatory response were examined after treatment with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) as 200 IU PMSG and 100 IU hCG, 300 IU PMSG and 150 IU hCG, or 1,500 IU PMSG only, followed by 100, 150 or 750 IU hCG 72 h later, respectively. The optimal protocol was determined to be the combination treatment of 200 IU PMSG and 100 IU hCG followed by 100 IU hCG. The administration of 200 IU PMSG and 100 IU hCG was effective in inducing estrus regardless of the strain, although there was a strain difference in the ovulatory response. These results indicate that treatment with a low-dose combination of PMSG and hCG provides one of the simplest methods for induction of estrus and ovulation in pubertal miniature pigs.     

Evaluation of systems for collection of porcine zygotes for DNA microinjection and transfer

Crossbred gilts and sows (n=116) were used for the collection of 1-cell zygotes for DNA microinjection and transfer. Retrospectively, estrus synchronization and superovulation schemes were evaluated to assess practicality for zygote collection. Four synchronization and superovulation procedures were used: 1) sows were observed for natural estrous behavior; 1000 IU human chorionic gonadotrophin (hCG) was administered at the onset of estrus (NAT); 2) cyclic gilts were synchronized with 17.6 mg altrenogest (ALT)/day for 15 to 19 days followed by superovulation with 1500 IU pregnant mares serum gonadotropin (PMSG) and 500 IU hCG (LALT); 3) gilts between 11 and 16 days of the estrous cycle received 17.6 mg ALT for 5 to 9 days and PMSG and hCG were used to induce superovulation (SALT); and 4) precocious ovulation was induced in prepubertal gilts with PMSG and hCG (PRE). A total of 505 DNA microinjected embryos transferred into 17 recipients produced 7 litters and 50 piglets, of which 8 were transgenic. The NAT sows had less (P < 0.05) ovarian activity than gilts synchronized and superovulated by all the other procedures. Synchronization treatments with PMSG did not differ (P > 0.05) in the number of corpora hemorrhagica or unovulated follicles, but SALT and PRE treaments had higher ovulation rates than LALT (24.7 +/- 2.9, 24.3 +/- 1.8 vs 11.6 +/- 2.7 ovulations; X +/- SEM). The SALT and PRE treatments yielded 12.3 +/- 2.6 and 17.7 +/- 1.7 zygotes. Successful transgenesis was accomplished with SALT and PRE procedures for estrus synchronization and superovulation.     

Effect of electrocautery of nonovulated day 1 follicles on subsequent morphological variation among day 11 porcine embryos

One hundred and thirteen crossbred gilts were used in three experiments to examine the relationship between the pattern or sequence of ovulation and subsequent variation in the morphology of Day 11 embryos. In the first experiment, the percentage of follicles that had ovulated was determined in individual gilts at 26, 30, 34, or 38 h after the onset of estrus (n = 20) and 39, 41, 43, 45, or 47 h post-injection of human chorionic gonadotropin (n = 25; hCG, 1000 IU). The second experiment consisted of observing the percentage of follicles ovulated in 52 additional gilts at 34 h after the onset of estrus (Day 0). In the third experiment, the morphological variation among littermate embryos was compared on Day 11 between sham-operated control gilts (n = 8) and gilts whose nonovulated follicles were destroyed by electrocautery (n = 8) on Day 1. Results of these experiments indicated that the pattern of ovulation in gilts was skewed (p less than 0.01). Ovulation, induced with hCG, appeared to occur in a majority of follicles during a short period of time, whereas the remaining ovulations occurred over a longer interval. Of the 57 gilts observed at 34 h after natural estrus, ovaries of 25 gilts contained corpora hemorrhagica (CH) and follicles; one gilt had 1 CH and 17 follicles, and 24 others had 10-17 CH with 1-4 follicles remaining. Destruction of these nonovulated follicles resulted in a more (p less than 0.01) uniform group of Day 11 embryos and with fewer (p less than 0.05) small embryos. These data demonstrated that the pattern of ovulation may affect morphological variation in embryonic development such that some of the later ovulating follicles may represent smaller embryos within a litter.     

Pregnancy in the domestic cat after vaginal or transcervical insemination with fresh and frozen semen

The objective was to compare pregnancy rates in domestic cats using fresh semen for intravaginal artificial insemination (IVI), either at the time of hCG treatment for induction of ovulation, or 28 h later, and to compare pregnancy rates following IVI or transcervical intrauterine insemination (IUI) of frozen–thawed semen. Eighteen queens were inseminated during 39 estrus cycles. Fresh semen with 13.5 ± 5.4 × 10⁶ sperm (range, 6.8–22 × 10⁶) collected by electroejaculation from four male cats was used in Experiment 1, and cryopreserved semen (20 × 10⁶ sperm, with 70 ± 5% post-thaw motility) from one male cat was used in Experiment 2. Serum concentrations of estradiol-17β and progesterone were determined in most queens on the day of AI and again 30–40 days later. Treatment with 100 IU of hCG 3 days after the onset of estrus induced ovulation in 95% of treated queens. Pregnancy rates to IVI with fresh semen at the time of hCG administration versus 28 h later were not different (P = 0.58); overall 33% (5/15) of the queens became pregnant. For frozen–thawed semen, AI was consistently done 28 h after hCG administration; IUI and IVI resulted in pregnancy rates of 41.7% (5/12), whereas no queen (0/12) became pregnant by IVI (P = 0.0083). In conclusion, an acceptable pregnancy rate was obtained with frozen–thawed semen in the domestic cat by non-surgical transcervical IUI; this method might also be useful in other small felids.     

Short synchronization system for estrus cycles in dairy heifers: a preliminary report

In previous studies we demonstrated that the administration of a luteolytic dose of cloprostenol to dairy cows in luteal phase, followed by hCG plus estradiol benzoate (EB) 12 h later, led to successful timed AI 48 h after the initiation of treatment. This article reports two consecutive studies. In Study 1 we determined the pregnancy rate of dairy heifers in luteal phase (established by palpation per rectum) treated with cloprostenol followed by 250 IU of hCG plus 1 mg of EB 12 h later, and inseminated 48 h after cloprostenol injection. Study 2 was designed to evaluate the efficiency this synchronization protocol, irrespective of the estrus stage of the animals. In Study 1, 1272 Friesian heifers aged 14 to 16 months with a palpable corpus luteum received 500 mcg cloprostenol. Heifers were then synchronized either according to the hCG plus EB protocol (hCG-EB, n=637), or by a second dose of cloprostenol 11 d later (PG, n=636). Animals in this last group served as controls and were inseminated 72 and 96 h after the second cloprostenol injection. The pregnancy rate was significantly higher (P<0.0001) in the hCG-EB group (59.5%, 379/637) than in PG (44.8%, 285/636). In Study 2, 135 contemporary heifers (with no corresponding information on estrus stage) were subjected to the same protocol as those in the hCG-EB group of Study 1. These animals were classified in retrospect according to estrus stage established by plasma progesterone concentration. Pregnancy rates were 66.7% (24/36), 51% (25/49) and 58% (29/50) for animals in the follicular, early/late luteal, and mid-luteal phase, respectively. The total pregnancy rate was 57.8% (78/135). These findings indicate an improved pregnancy rate for heifers subjected to single insemination after cloprostenol/hCG/EB synchronization, compared to double insemination after synchronization by 2 cloprostenol injections 11 d apart. The cloprostenol/hCG/EB protocol did result in acceptable pregnancy rates after timed AI of dairy heifers regardless of their estrus cycle phase.     

Developmental competence of domestic cat follicular oocytes after fertilization in vitro

Empirical evaluation of variables affecting oocyte collection, in vitro fertilization, and embryo transfer resulted in establishing a successful procedure for the artificial production of offspring in the domestic cat. Female cats were treated with pregnant mare's serum gonadotropin (PMSG, 150 IU) followed 72 or 80 h later with 100 or 200 IU human chorionic gonadotropin (hCG). After laparoscopic collection, follicular oocytes were inseminated in vitro with ejaculated, processed spermatozoa, cultured (37 degrees C, 5% CO2), and then examined for evidence of fertilization. Two- to 4-cell stage embryos were transferred to the oviducts of oocyte donors. Oocyte donor cats and naturally mated controls also were subjected to sequential laparoscopic examinations and blood sampling to assess corpora lutea (CL) function. At 24-30 h of culture, fewer (p less than 0.001) degenerate oocytes were observed in cats receiving 100 IU hCG (8.2%) compared to those receiving 200 IU (20.6%), regardless of the PMSG-hCG interval. Overall fertilization (48.1%) and cleavage (45.2%, at 30 h post-insemination) rates were greatest following an 80-h PMSG-hCG interval combined with the 100 IU hCG dose. Five of the 6 cats receiving 6 to 18 embryos became pregnant and produced from 1 to 4 kittens/litter. Gonadotropin-treated females subjected to follicular aspiration produced morphologically normal CL and circulating progesterone patterns that were qualitatively similar (p greater than 0.05) to control cats. These data indicate that domestic cat follicular oocytes are capable of fertilization in vitro, but success is dependent on both the timing and dose of the hCG stimulus. Follicles subjected to aspiration appear capable of forming normal, functional CL and the birth of live young after embryo transfer unequivocally demonstrates, for the first time, the developmental competence of in vitro-fertilized carnivore oocytes.     

Importance of estrus on pregnancy per insemination in suckled Bos indicus cows submitted to estradiol/progesterone-based timed insemination protocols

The objective was to evaluate the effect of estrus occurrence (based on removal of tail-head marks) on ovarian responses and pregnancy per AI (P/AI; 30 d after AI) in suckled Bos indicus beef cows submitted to timed AI (TAI) protocols. Cows received an intravaginal device containing 1.0 g progesterone, and 2.0 mg estradiol benzoate im; 8 d later, the intravaginal device was removed, and they were given PGF_2α (0.25 mg of cloprostenol sodium) and 300 IU of eCG, with TAI 48 to 52 h later. In Experiment 1, cows were assigned to receive one of three treatments: 1 mg of estradiol cypionate (ECP) im at progesterone (P4) device removal (N = 178); 10 μg of GnRH im at TAI (N = 190); or both treatments (N = 172). In cows given estradiol (ECP or ECP + GnRH), more displayed estrus (P = 0.002) and became pregnant (P < 0.0001) compared with those receiving only GnRH. In Experiment 2, the effect of the occurrence of estrus on ovarian responses was evaluated in cows (N = 53) synchronized using ECP at device removal. Cows that displayed estrus had a greater diameter of the largest follicle (LF) at device removal (P < 0.0001), a greater diameter at TAI (P < 0.0001), a greater ovulation rate (P = 0.02), a larger CL (P = 0.02), and a greater P4 concentration (P < 0.0001) than cows that did not display estrus. In Experiment 3, the effect of GnRH treatment on P/AI at TAI was evaluated in cows that received ECP at device removal, and either displayed, or did not display, estrus (N = 726). There was no estrus by GnRH interaction (P = 0.22); the P/AI was greater (P < 0.0001) in cows that displayed estrus (61.9%) than cows that did not display estrus (41.4%). However, GnRH did not improve (P = 0.81) P/AI (GnRH = 53.7% vs. no GnRH = 52.6%). In conclusion, exogenous estradiol at device removal increased both the proportion of suckled Bos indicus cows that displayed estrus and P/AI. Cows that displayed estrus had better ovarian responses (i.e., larger follicles at TAI, a greater ovulation rate, larger CL, and greater P4 concentrations) following an estradiol/P4-based synchronization protocol. Although occurrence of estrus improved pregnancy outcomes, GnRH at TAI did not improve P/AI in suckled Bos indicus cows treated with ECP, regardless of estrus occurrence.     

Artificial insemination with cryopreserved sperm from feline epididymides stored at 4 °C

Recovering and storing sperm from the epididymides of males of rare felidae is useful for preserving the species. The objective of the present study was to determine pregnancy rates following artificial insemination (AI) of frozen-thawed epididymal sperm, which were cryopreserved following low-temperature storage of the epididymides. In this study, these sperm were used for unilateral intrauterine AI (UIUAI) or unilateral intratubal AI (UITAI) using 40 × 10⁶ and 10 × 10⁶ sperm, respectively. The caudal epididymides of 17 cats were stored at 4 °C for 24 h after castration. Artificial insemination of seven female cats was performed on Days 3 or 4 (start of estrus = Day 1) by UIUAI, 20 h after injection of 100 IU hCG to induce ovulation. Furthermore, UITAI at 24 h (UITAI-24) or 30 h (UITAI-30) after hCG were also done (five cats per group). It was noteworthy that AI by UIUAI and UITAI-24 was performed before ovulation, whereas AI by UITAI-30 was performed after ovulation. Pregnancy rates were 28.6% (2/7) by UIUAI, 80% (4/5) by UITAI-24, and 20% (1/5) by UITAI-30. Litter size was one or two by UIUAI, and one to four by UITAI. Spontaneous abortion occurred on Days 25-30 of pregnancy in one of the two female cats pregnant following UIUAI, and in two of five female cats pregnant following UITAI. Based on the high pregnancy rate obtained with 10 × 10⁶ sperm in the UITAI-24 group (AI performed before ovulation), we concluded that this was the most appropriate method for AI with frozen-thawed epididymal sperm after initial low-temperature storage of epididymides.     

The impact of dose of FSH (Folltropin) containing LH (Lutropin) on follicular development, estrus and ovulation responses in prepubertal gilts

FSH is favored over chorionic gonadotropins for induction of estrus in various species, yet little data are available for its effects on follicle development and fertility for use in pigs. For Experiment 1, prepubertal gilts (n = 36) received saline, 100 mg FSH, or FSH with 0.5 mg LH. Treatments were divided into six injections given every 8 h on Days 0 and 1. Proportions of gilts developing medium follicles were increased for FSH and FSH-LH (P < 0.05) compared to saline, but follicles were not sustained and fewer hormone-treated gilts developed large follicles (P < 0.05). No gilts expressed estrus and few ovulated. Experiment 2 tested FSH preparations with greater LH content. Prepubertal gilts (n = 56) received saline, FSH-hCG (100 mg FSH with 200 IU hCG), FSH-LH5 (FSH with 5 mg LH), FSH-LH10 (FSH with 10 mg LH), or FSH-LH20 (FSH with 20 mg LH). FSH-LH was administered as previously described, while 100 IU of hCG was given at 0 h and 24 h. Hormone treated gilts showed increased (P < 0.05) medium and large follicle development, estrus (>70%), ovulation (100%), and ovulation rate (>30 CL) compared to saline. There was an increase (P < 0.05) in the proportion of hormone-treated gilts with follicular cysts at Day 5, but these did not persist to Day 22. These gilts also showed an increase in poorly formed CL (P < 0.05). FSH alone or with small amounts of LH can induce medium follicle growth but greater amounts of LH at the same time is needed to sustain medium follicles, stimulate development of large follicles and induce estrus and ovulation in prepubertal gilts.     

Induction of ovarian follicular cysts in the pregnant rat by human chorionic gonadotropin.

Prolonged stimulation by human chorionic gonadotropin (hCG) induces ovarian follicular cysts in progesterone-synchronized immature rats [Bogovich, Endocrinology 1989; 124:1646-1653]. To determine if unabated stimulation by hCG has a similar effect on follicular development in adult ovaries, pregnant rats were given either 0 (control), 1, or 3 IU hCG twice daily for 9 days beginning on Day 13 of pregnancy. By Day 22 of pregnancy, rats treated with 1 IU hCG possessed large antral follicles at least 1 mm in diameter: approximately 33% larger than the diameters of preovulatory follicles observed in control rats (0 IU hCG). In contrast, rats treated with 3 IU hCG displayed ovarian follicular cysts up to 5 mm in diameter, with well-developed thecae and just a remnant of granulosa cells. Progesterone, androstenedione, and estradiol accumulation was greater in follicular incubates from hCG-treated rats than in incubates from control rats. Progesterone increased in response to cAMP in incubates from all treatment groups on all days tested. Androstenedione increased in response to cAMP on Day 22 of pregnancy for follicles from control animals, on all days tested for follicles from rats treated with 1 IU hCG, and on Days 15-19 for follicles from rats treated with 3 IU hCG. Androstenedione production in the presence of 300 ng of exogenous testosterone was significantly greater in follicular incubates from animals treated with 1 and 3 IU hCG than incubates from control animals on Days 19-22 of pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)