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Contemporary systems for oocyte retrieval and culture of both cattle and human embryos are suboptimal with respect to pregnancy outcomes following transfer. In humans, chromosome abnormalities are the leading cause of early pregnancy loss in assisted reproduction. Consequently, pre-implantation genetic testing for aneuploidy (PGT-A) is widespread and there is considerable interest in its application to identify suitable cattle IVP embryos for transfer. Here we report on the nature and extent of chromosomal abnormalities following transvaginal follicular aspiration (OPU) and IVP in cattle. Nine sexually mature Holstein heifers underwent nine sequential cycles of OPU-IVP (six non-stimulated and three stimulated cycles), generating 459 blastocysts from 783 oocytes. We adopted a SNP-array approach normally employed in genomic evaluations but reanalysed (Turner et al., 2019; Theriogenology125: 249) to detect levels of meiotic aneuploidy. Specifically, we asked whether ovarian stimulation increased the level of aneuploidy in either trophectoderm (TE) or inner-cell mass (ICM) lineages of blastocysts generated from OPU-IVP cycles. The proportion of Day 8 blastocysts of inseminated was greater (P, Highlights • In vitro oocyte maturation rather than ovarian stimulation contributes to aneuploidy. • Trophectoderm biopsies accurately represent the ploidy status of the overall embryo. • SNP-array analyses facilitate simultaneous genomic evaluation and aneuploidy screening. • Incidence of aneuploidy declines in developmentally more advanced embryos. • High degree of variability in the incidence of aneuploidy exists between donors.

The objective of ovarian superstimulatory treatments in cattle is to obtain the maximum number of viable embryos by stimulating growth of antral follicles and ovulation of competent oocytes. While factors inherent to the donor animal are critical, an increased knowledge of ovarian physiology, gonadotropin biochemistry and the ability to manipulate ovarian function have provided alternatives for the design of simple and successful protocols for superovulation in cattle. Recent protocols have also been made more user-friendly and allowed for the grouping of donors for successful superovulation. Although the number of reports associating FSH profiles with superovulatory response is limited, studies designed to reduce the number of FSH treatments necessary to induce superstimulation may provide guidance for the development of optimized gonadotropin treatment protocols. Although high peak levels of circulating FSH following a single administration of Folltropin-V have been shown to be associated with a reduced superstimulatory response, the ideal treatment protocol would seem to be to increase circulating FSH levels to values comparable to those required for the induction of follicle wave emergence, and to maintain these levels for at least 72 h (or 36 h for superstimulation prior to ovum pick-up) to allow follicles to reach an ovulatory size and acquire the capacity to ovulate.

The objective of the present study was to evaluate the effect of equine chorionic gonadotropin (eCG) administration associated to different proestrus lengths for Fixed-time AI (FTAI) in beef heifers. In Experiment 1, pre-pubertal heifers (n = 46) received a 6-day estradiol/progesterone-based treatment (J-Synch protocol), and were then allocated into four experimental groups in a 2 × 2 factorial design, to receive or not receive eCG (300 IU) at the time of intravaginal progesterone device removal, and to receive GnRH at 48 h or 72 h after device removal (to induce shortened and prolonged proestrus length, respectively). Endometrial samples were obtained 6 d after ovulation from the cranial portion of the uterine horn. The eCG administration induced greater serum estradiol-17β concentrations before ovulation (P 0.05) and greater proportion of heifers bearing a competent corpus luteum after ovulation (P = 0.054). Delaying GnRH administration from 48 h to 72 h induced a longer interval from device removal to ovulation (i.e., prolonged proestrus; P 0.05), larger diameter of the ovulatory follicle, and greater progesterone concentrations on Day 10-11 after ovulation. Heifers in eCG + GnRH72h group had more uterine receptors in luminal epithelium than those in eCG + GnRH48h group (PR and ERα), and than those in No eCG + GnRH72h group (PR) (P 0.05). No effect of eCG or GnRH treatments was found in endometrial gene expression of progesterone and estrogen receptors. In Experiment 2, a total of 2,598 heifers received the J-Synch protocol associated or not with eCG administration at device removal, followed by FTAI/GnRH at 60 or 72 h after device removal (i.e., prolonged proestrus protocol). Heifers that received eCG had greater P/AI than those not receiving eCG (P 0.05) and there was an interaction between eCG treatment and time of FTAI. The lowest P/AI was found in those heifers that received FTAI/GnRH at 72 h without eCG treatment at device removal (P 0.05), and no differences were found between the other experimental groups. In conclusion, prolonging the length of proestrus in J-Synch protocol improves ovulatory follicular diameter and luteal function; and the administration of eCG at device removal improves preovulatory estradiol concentrations and luteal function. Finally, P/AI was enhanced by eCG treatment and the improvement was more evident when FTAI/GnRH was performed at 72 h after device removal.

Objectives were to 1) characterize fixed-time AI (FTAI) pregnancy rates using the 5-Day CO-Synch + CIDR protocol in mature, suckled Bos indicus-influenced beef cows, 2) compare FTAI pregnancy rates in the latter to a modified version (5-Day Bee Synch + CIDR; Bee Synch I) that included treatment with prostaglandin F2α (PGF2α) at CIDR insertion on Day 0, and 3) test the hypothesis that elimination of both GnRH-1 at the onset of synchronization and the double dose of PGF on Day 5 (Bee Synch II) would not reduce FTAI pregnancy rates compared to Bee Synch I. For Experiment 1-trial 1, Brahman x Hereford (F-1) cows (n = 168) at least 40 d postpartum (PP; r = 40-92 d) at the time of CIDR insertion were administered the 5-Day CO-Synch + CIDR protocol with FTAI at 72 h after CIDR removal. Pregnancy rates to FTAI averaged 34.9 ± 1.9%. In Experiment 1-trial 2, fall- and spring-breeding Brahman x Hereford (F-1) beef cows (n = 269) were stratified by days PP and assigned randomly to receive either the 5-Day CO-Synch + CIDR (n = 136) or Bee Synch I (n = 133) protocol, with FTAI at 66 h after CIDR removal. Pregnancy rate to FTAI was greater (P 0.05) in Bee Synch I (52.6 ± 0.9%) than in the 5-Day CO-Synch + CIDR procedure (40.4 ± 5.7%). For Experiment 2, 422 mature Braford, Brangus, Nelore x Brahman, and Brahman crossbred cows (Bos indicus proportion unknown) at 4 locations were treated with Bee Synch I, with FTAI at 66 h. Overall FTAI pregnancy rate averaged 51.7 ± 2.1%. Finally, from 2013 through spring 2018, we used a switchback design using fall- and spring-breeding herds to compare Bee Synch I (402 observations) to Bee Synch II (393 observations). Overall frequency of detected estrus at 66 h using ESTROTECT™ breeding indicator patches was 57.2 ± 2.4%, conception rates of those detected in estrus was 64.4 ± 3.5%, and FTAI pregnancy rates averaged 52.3 ± 2.4%, none of which differed between treatments. Moreover, pregnancy rates to FTAI in both treatments did not differ in cows synchronized between 40 and 80 d PP but increased after 80 d PP (P 0.05). Bee Synch II, which eliminates GnRH-1 and the double dose of PGF2α on Day 5, results in FTAI pregnancy rates essentially identical to Bee Synch I but reduces synchronization costs and avoids the need for off-label (double dose PGF2α) drug use.

This study compared the outcome of two new timed artificial insemination (TAI) protocols in a milk-producing donkey farm. Ninety Amiata jennies were inseminated at the moment of ovulation induction with hCG, with fresh-transported semen that had been stored at room temperature from 3 up to 6 h, with an approximate average storage time of 4 h and a half. In both protocols, on Day 0 jennies were treated with alfaprostol (PGF2α), and on Day 7 they were checked by ultrasound (US) and, if in estrus, they were treated in order to induce ovulation and were then artificially inseminated. In the slow-short TAI protocol, jennies not already inseminated were treated again with PGF2α at Day 14. On day 21 US was repeated and estrus jennies were induced to ovulate and inseminated. In the fast-long TAI protocol, US was performed once a week in jennies not already inseminated and if found in estrus, they were induced to ovulate and inseminated, while those not in estrus were treated again with PGF2α. This protocol was repeated for up to nine weeks. The rates of inseminated/treated, pregnant/inseminated and pregnant/treated jennies were 76%, 56% and 43% for the slow-short TAI protocol and 94%, 47% and 44% for the fast-long TAI protocol. The age class and the lactation status of the jennies had no significant effect on synchronization success or final pregnancy rate. This study demonstrates that it is possible to achieve reasonable pregnancy rates through simplified TAI protocols in jennies, reducing animal handling to a minimum.

Our goal was to develop an objective computer-assisted volumetric method of assessing vascular flow from colour Doppler ultrasound data of ovarian structures recorded by free-hand movement. We hypothesized that a vascularity index (ratio of the region of blood flood to the region of ovarian structure) obtained from the three-dimensional volumetric analysis would be more precise (less variable) than conventional two-dimensional analysis of single images in estimating the functional status of the preovulatory follicles and corpus luteum. Doppler ultrasound cineloops of water buffaloes (Bubalus bubalis; n = 22) ovaries were recorded daily from 12 h before GnRH treatment to four days after ovulation. Cineloops were processed using Fiji and Imaris software packages for segmenting the area (two-dimensional analysis) and the volume (three-dimensional analysis) occupied by the blood-flow and associated tissue to calculate the vascularity index. For volumetric measurement, all images in a cineloop were used (i.e., no a-priori selection of images) while for two-dimensional analysis, three images from the region with apparent maximum vascularity were selected. The volumetric method was verified with theoretical ellipsoidal volume of the follicle (r = 0.96 P 0.01) or corpus luteum (r = 0.58 P = 0.02). The variability in the follicular vascularity index among animals was lower using the volumetric method than two-dimensional analysis (0.018 ± 0.002 vs 0.030 ± 0.005, P 0.01), while the variability for CL vascularity was similar between methods (P = 0.23). An increase in the follicular vascularity index was detected at 12 h after GnRH treatment using both methods (two-dimensional: 0.030 ± 0.008, P 0.01; three-dimensional: 0.016 ± 0.006, P 0.02). Buffaloes that ovulated tended to have a greater increase in 3D vascularity index than non-responding buffaloes (P = 0.06); the two-dimensional method was not able to detect these changes. Using the three-dimensional method, a moderate positive correlation (r = 0.59; P = 0.02) was evident between the follicular vascularity index at 14-16 h after GnRH treatment and follicular diameter. In conclusion, an objective volumetric method for assessing relative ovarian blood flow changes was developed using Doppler ultrasound cineloops recorded by free-hand movement. The 3-dimensional method eliminates the need for a-priori selection of images and is more precise as a result of decreased technical variability.

Made available in DSpace on 2019-10-06T17:13:02Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-09-15 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The aim of the present study was to evaluate the efficacy of a GnRH analog for induction of ovulation in Brazilian Northeastern jennies (Equus asinus) with different follicle diameters. Four consecutive estrus of 10 jennies were used in a crossover study; C (Control, n = 10) jennies were evaluated by transrectal palpation and ultrasonography until a spontaneous ovulation and the intervals between the predetermined follicular size (25–28 mm [C1], 29–32 mm [C2] and 33–36 mm [C3] follicle) and ovulation were registered. In treated cycle, jennies had the ovulation induced by 250 μg of Histrelin acetate (Strelin®, Botupharma, Botucatu, Brazil) when respective follicle diameters 25–28 mm (T1), 29–32 mm (T2) and 33–36 mm (T3) were diagnosed. Ovulation was monitored by transrectal palpation and ultrasonography. Different follicle diameters significantly affected (P < 0.05) the interval until ovulation between control and matched treated cycles. Interval between prostaglandin administration and ovulation diagnosis was lower in jennies from T2 group (145.2 ± 34.6 h) compared with the control cycle (220.0 ± 41.8 h) and also with other treated cycles (T1 - 209.8 ± 48.0 h; T3 – 183.3 ± 33.9 h). Histrelin acetate treatment also reduces the interval between detection of predetermined follicular size and ovulation (P < 0.05) in all treated cycles groups compared with matched control group. Higher percentage (P < 0.05) of jennies had success of ovulation induction (36–48 h after Histrelin acetate injection) in all treated cycles in contrast with the matched control group. In addition, in comparison among treated cycle groups, more (P < 0.05) jennies (100%) in T2 ovulated between 36 and 48 h after ovulation induction, compared with T1 and T3, which did not differ (P > 0.05) from each other. Edema scoring and ovulation were not associated events (r = 0.0219). In conclusion, jennies with 29–32 mm follicles satisfactory responded to ovulation induction with Histrelin acetate, which allowed the shortening of interovulatory interval in all groups evaluated. Sao Paulo State University (UNESP) School of Veterinary Medicine and Animal Science Federal University of Bahia (UFBA) Sao Paulo State University (UNESP) School of Veterinary Medicine and Animal Science

The present study evaluated follicular superstimulatory (FSS) and superovulatory (SOV) responses and in vivo embryo production in lactating Simmental cows treated with FSH starting 1 or 2 days after follicle aspiration (FA). The performance of a lengthened superovulation program, named 6dFSH-P36-hCG60, is described. At random stages of the estrous cycle, cows (n = 52) were subjected to ultrasound-guided transvaginal aspiration of all follicles ≥ 5 mm. After FA, cows were randomly assigned to one of two groups in which FSH treatments started 1 or 2 days after FA (groups FA-1D and FA-2D, respectively). Cows were superstimulated with a total of 500 μg pFSH over 6 days on a decreasing dose schedule and were pre-treated with a single dose of 400 IU of eCG 24 h before the start of FSH treatments. Follicular superstimulatory (the mean numbers of follicles ≥ 8 mm on the day of hCG treatment) and SOV responses (the mean numbers of CL and cows with ≥ 3 CL at the time of collection) were similar in FA-1D and FA-2D groups. However, when compared to FA-1D group, the number of unfertilized ova tended to decrease (0.4 vs 1.7; P = 0.065) and percentage of fertilized ova tended to increase (95.8% vs 84.6%; P = 0.066) in FA-2D group. Moreover, the mean numbers and percentages of both transferable embryos (8.0 and 77.6% vs 6.4 and 57.7%) and freezable embryos (5.3 and 51.5% vs 3.5 and 31.1%) were numerically higher in FA-2D group than FA-1D group. The results of the study suggest that starting a lengthened superovulation programs in Simmental cows 2 days after FA has potential to increase percentage of fertilized ova and the number of transferable and freezable embryos, although new studies may be needed to confirm this findings.

Many mares are susceptible to persistent mating-induced endometritis (PMIE), an important cause of reduced fertility. Platelet lysate (PL) derives from freeze-thawing platelets after concentration, so that growth factors are released from the platelets. Among the advantages of PL compared to platelet-rich plasma (PRP), it can be frozen stored and allogenic use for PL might also be conceivable. Platelet-rich plasma beneficially reduced inflammatory response in PMIE mares when administered 24 h pre- or 4 h post-AI. The aim of this study was to test the effect of PL on inflammatory uterine response in mares susceptible to PMIE. A total of 14 mares susceptible to PMIE (based on presence of fluid or inflammatory cells 24 h after AI) underwent an untreated (Ctr) cycle followed by a treated (PL) cycle. From each mare, 100 mL of citrated whole blood was obtained for PRP production by centrifugation. The resultant PRP was brought to a final volume of 10 mL with platelet poor plasma and frozen at −80 °C to obtain PL. On untreated cycles, mares were inseminated with frozen-thawed semen 36 h after ovulation induction. On treated cycles, PL was thawed, infused into the uterus 12 h after ovulation induction, and AIs were performed 24 h later. The number of neutrophils in uterine cytology (score 1(normal)-3(severe inflammation)) evaluated by optical microscopy, uterine fluid accumulation (height x width) and uterine edema (score 0–3) observed in ultrasonography, were analysed. Pregnancy was evaluated by ultrasonography 14 days after ovulation. A significant decrease (P < 0.05) was observed on cytology score (PL 1.3 ± 0.1 vs Ctr 2.0 ± 0.1), fluid accumulation (PL 79.5 ± 30.1 mm2 vs Ctr 342.7 ± 52.9 mm2) and edema score (PL 1.8 ± 0.2 vs Ctr 2.3 ± 0.2) in treated mares. Pregnancy rate in PL-treated cycles (3/12) and control cycles (2/14), were not significantly different (P > 0.05). According to the results, we conclude that treatment with PL in mares classified as susceptible to PMIE appears to reduce the inflammatory response after breeding, based on clinical signs of uterine edema, IUF accumulation and PMNs migration.

On commercial farms, rabbit does are subjected to a reproductive rhythm that does not account for their welfare or physiology, leading to reduced longevity and consequently high annual replacement. The European Food Safety Authority (EFSA) recommends limited and infrequent use of hormone treatments, and suggests replacement with alternative methods that do not threaten animal welfare when possible. In the present study, we aimed to determine whether the GnRH analogue lecirelin acetate could be administered by inclusion in the seminal dose during insemination. Twenty 9-month-old does (Grigio del Monferrato, autochthonous Italian breed),each having two previous deliveries, were individually housed and divided into two groups at artificial insemination. The control group received 0.2 mL of intramuscular lecirelin (Dalmarelin, Fatro®, Italy) prior to insemination. The intravaginal group was inseminated with a seminal dose that included 0.3 mL Dalmarelin. The experiment was performed for six consecutive reproductive cycles at 42-day intervals, and included a total of 120 inseminations. Prior to each insemination, the heterospermic pooled semen samples were assessed for sperm motility and morphology. Each ejaculate was divided into two samples, with and without lecirelin addition. Compared to the control group, the does with intravaginal Dalmarelin administration showed equal or greater sexual receptivity, which resulted in a higher fertility rate over increasing cycles. The seminal dose volume was very low, possibly explaining the better results in the intravaginal group, which received a similar amount of hormone (0.3 mL/doe) as the control group (0.2 mL/doe). The negative performance of the control group may have also been due to anti-GnRH formation, and the more stressful method of ovulation induction. The number of live-born kits did not significantly differ between groups. Progressive motility was significantly positively correlated with motility characteristics, including VAP, VSL, ALH, BCF, STR, and LIN. Overall, our present findings supported that the incorporation of GnRH in a seminal dose could be used for ovulation induction in rabbit does. Further studies should identify the optimal dose of GnRH for intravaginal administration, taking into account that the intravaginal absorption capacity is about 10 times smaller than the intramuscular absorption capacity.

A recombinant long-acting ovine follicle stimulating hormone (roFSH) has been devised and its biological effectiveness following a single dose has been assessed in several experiments under field conditions, in pasture-based beef and dairy farming in New Zealand. Owing to the molecular structure of this long-acting roFSH, which includes additional N-glycosylation sites, a single dose combined with a simple CIDR-based superovulatory regime elicits successful ovarian stimulation with averages of 11.8 corpora lutea and of 6.1 good quality embryos collected in cattle. Solid performance of this novel FSH was demonstrated in several beef and dairy breeds which included yearling heifers and mixed age cows, with embryo production results in the same range as those observed nowadays with eight doses of commercial pituitary FSH (pFSH). Viable embryos produced from these collections, when implanted either fresh or frozen and thawed, gave pregnancy rates in recipients similar to those collected from cows and heifers superstimulated with pFSH. Repeated superovulatory treatment of the same cows was not associated with a decrease in ovarian response or embryo yield. The single administration of this long-acting roFSH when combined with a modified simple superovulatory regime has the advantage of reducing animal welfare concerns, lowering labour resource requirements and giving similar results to other commercially prepared pituitary FSH extracts.

This study was conducted to develop simple superovulation protocols for dromedary camels using eCG. In experiment 1, camels received either 1000, 2000, 3000, 4000, 5000 or 6000 IU eCG. In experiment 2, camels received either 400 mg FSH (Folltropin-V) twice-daily over 5 days or 3000 IU eCG. In experiment 3, camels received 3000 IU eCG either at 2, 3, 4 or 5 days after ovulation induction. Ovarian response and embryo yield were evaluated in all experiments and embryos collected from camels treated with FSH and eCG were transferred to recipients to examine pregnancy rates. The mean number of ovulations (12.6 ± 1.5 and 13.3 ± 1.2 vs 3.4 ± 0.3, 6.2 ± 0.6 and 9.3 ± 1.0, respectively) and transferable embryos (4.6 ± 1.3 and 4.8 ± 1.0 vs 1.6 ± 0.2, 2.2 ± 0.4 and 1.1 ± 0.4, respectively) with 3000 and 4000 IU eCG doses were higher compared to 1000, 2000 and 6000 IU eCG doses (P 0.05). Doses of 5000 and 6000 IU eCG resulted in a higher number of unovulatory follicles than other doses (P 0.05). The FSH treatment resulted in higher number of ovulatory follicles (21.8 ± 1.3 vs 14.8 ± 1.7) and ovulations (18.5 ± 1.1 vs 13.9 ± 1.4) compared to eCG (P 0.05). However, the number of transferable embryos and pregnancy rates were similar in these treatments. The timing of eCG treatment after ovulation induction did not affect the number of ovulatory follicles and transferable embryos but eCG treatment at 5 days after ovulation induction reduced the number of ovulations (P 0.05). In conclusion, the optimal dose of eCG to induce superovulation is 3000-4000 IU and it produces a comparable embryo yield to FSH, and can be administered at 2-4 days after ovulation induction.

The objective of the current study was to investigate the effect of subluteal progesterone environment on the efficacy of the ovarian responses in goats passively immunized against endogenous inhibin. Twelve cycling female Shiba goats were synchronized using two doses of prostaglandin F2α 11 days apart. After detection of ovulation (D0, by ultrasonography), goats were randomly assigned into 2 groups: (1) Subluteal P4 group (SLP; n = 6), treated with a previously once-used controlled intravaginal progesterone-releasing device (ou-CIDR) intravaginal from D 6 to D 12 concurrently with a luteolytic dose of PGF2α at D6 (2) Normal Luteal P4 group (NLP; n = 6), had neither ou-CIDR nor PGF2α. In both groups, goats were treated with an intravenous injection of 10 ml of inhibin antiserum at D10. After that, another luteolytic dose of PGF2α was administrated to goats in both groups at D12, and withdrawal of ou-CIDR in the SLP group. Detection of estrous behavior was checked again 24 h following PGF2α administration and or ou-CIDR withdrawal at 8 h intervals with a teaser buck. The follicle populations ≥4 mm in each goat were monitored through transrectal ultrasonography daily from 1 day after inhibin antiserum administration (0 h) and continued until ovulation had been detected (disappearance of large dominant follicles by ultrasonography). In addition, a repeated ultrasonographic scanning was performed 7 days after ovulation, and the ovulation rate had been estimated by counting the number of observed corpora lutea by ultrasonography. Blood samples were collected for measurement of plasma circulating inhibin binding activity, FSH, LH, estradiol (E2), and progesterone (P4) in both groups. Results revealed significant increases in maximum diameter of the preovulatory follicles (5.92 ± 0.17 mm vs 5.31 ± 0.26 mm; P 0.05), and the mean maximum number of follicles ≥ 4 mm in diameter (18.45 ± 3.40 vs 12.15 ± 6.21; P 0.05) in the SLP group compared with that of the NPL group. No significant differences were observed between both groups in term of inhibin binding activity %, and FSH. Following immunization, significant increases in the concentrations of circulating LH (at 72 h) and E2 (from 48 to 84 h) were noted in the SLP group as compared with the values obtained from the goats in the NLP group. Moreover, significant increases in ovulation rate (13.85 ± 1.40 vs 5.67 ± 2.10; P 0.01) and subsequent P4 concentrations (from 192 to 288 h) were found in the SLP group as against those of the NLP group. In conclusion, maintaining a subluteal P4 environment concurrently with passive immunization against endogenous inhibin may improve the superovulatory response in female Shiba goats.

Endocrine effects as 11-ketotestosterone (11-KT), an unaromatizable androgen, regulating the follicles growth in the previtellogenic stage of eel reproduction have been widely elucidated. However, the influence of aromatizable androgens on the brain-pituitary-gonad axis during oogenesis in A. japonica has not been clearly elaborated. In the study, androstenedione (AD) and 17α-methyltestosterone (MT) were employed together to induce ovary development of seven-year-old female Anguilla japonica through feeding or exposure in the migration season. After female A. japonica had been fed with commercial diet containing 5 mg AD and MT kg d−1 body weight respectively for 45 d in fresh water (Trial I), the development of oocytes still remained at the oil droplet stage, but the GSI and follicle diameter increased significantly. The serum 11-KT level and expression of liver vitellogenin mRNA were significantly elevated. After female fish had been exposed to seawater containing 50 μg L−1 AD and MT respectively for 45 d (Trial II), the ovaries of A. japonica almost reached midvitellogenic stage and the GSI and follicle diameter increased significantly. Yolk granular layer was observed in the peripheral ooplasm. The serum 11-KT level maintained consistently low, and the serum E2 level declined significantly to a relatively low level. The expression levels of ovarian arα and cyp19a1, brain (with pituitary together) mGnRH and lhβ increased significantly. The results showed that A. japonica in Trial II appeared a higher ovarian development than those in Trial I. These findings indicated that AD and MT increased the oil droplet and enlarged follicle diameter in previtellogenic stage, while the vitellogenesis and gonadotropin release did not occur in Trial I. In Trial II, AD and MT promoted vitellogenesis by stimulating the ovary expression of arα and by up-regulating brain mGnRH and pituitary lhβ expression.

A novel gel formulation was selected for intravaginal delivery of the GnRH agonist (triptorelin) for synchronizing ovulation in pigs. Studies with gilt models were used to assess LH response profiles. The lowest dose of triptorelin that induced the most gilts to show an LH surge was 100 μg in 1.2% methylcellulose gel. This formulation had a similar effect in weaned sows while also advancing ovulation. The timing of administration was evaluated in sows after weaning. Administration at 96 h induced more sows to ovulate (58%) by 48 h compared to treatment at estrus (45%) or for controls (34%), but the desired level of ovulation synchrony was not achieved. As a result, greater doses of triptorelin were tested and 200 μg given at 96 h after weaning, induced 81% of sows to ovulate within 48 h after treatment. The best synchrony of ovulation occurred when given at 96 h after weaning compared to earlier or later intervals. The optimum time to give a single fixed time AI (SFT-AI) after administration of 200 μg of triptorelin in 1.2% gel (OvuGel®) at 96 h after weaning was tested. A SFT-AI at 22 ± 2 h after OvuGel achieved the highest fertility and was practical for staff during the normal work day. In field trials, a SFT-AI 22 ± 2 h after all weaned sows were treated with OvuGel improved (P = 0.04) farrowing rate to 82.5% compared to control sows weaned (80.1%), with no effect on numbers of pigs born alive (12.1). Research continues for identifying the advantages for use of OvuGel in different production systems, and potential application for use in gilts.

Assisted reproduction technologies are widely used in humans and domestic animals and often include follicle stimulating hormone (FSH) in the protocol. One limitation with most of the available FSH preparations is the relative short half-life in the circulation that dictates multiple daily injections for the desired follicle development and superovulation. The development of bioactive long-acting structurally modified FSH analogs is desirable for human and veterinary use. In addition, optimal preparations and/or formulations are expected to improve the regimen and efficiency of the treatment. This review briefly describes the approaches that have been explored to extend the half-life of FSH in the circulation. These include strategies to increase the mass and/or charge of FSH and to prevent the dissociation of the hormone to inactive subunits components. Most of these strategies, except one that led to a registered drug (Elonva) indicated for controlled ovarian stimulation protocols in humans, are still in experimental stage.

A single dose protocol of FSH for superstimulation in cattle may improve compliance and superovulatory response. A single subcutaneous (sc) administration of pFSH was efficacious, but response depended on body condition and injection site; the adipose tissue pad behind the shoulder was most efficacious. Inconsistent results in Holsteins were partially overcome by sc administration of 75% of the total pFSH dose behind the shoulder on the first day followed by 25% 48 h later. An alternative would be to combine FSH with polymers that cause it to be released slowly over several days. Hyaluronan is found normally in most animal tissues and is nonreactive when administered parentally. A single intramuscular (im) administration of pFSH in a 2.0% hyaluronan induced a superovulatory response that did not differ from twice daily im administration over 4 d. However, 2.0% hyaluronan was viscous and difficult to mix with FSH. Although solutions of 1.0 and 0.5% hyaluronan were less viscous, they lacked efficacy as a single im administration. However, superovulatory response was high when either 1.0 or 0.5% hyaluronan was used in a two-dose im protocol; two-thirds on the first day and one-third 48 h later. A single im administration of FSH in 0.5% hyaluronan effectively induced superstimulation for OPU in cattle. Successful superovulation in the cow was associated with circulating FSH levels that were similar to endogenous FSH levels prior to follicular wave emergence; however, levels must be maintained above baseline for at least 72 h, or 36 h for OPU. Circulating FSH levels following a single sc administration of 400 mg NIH-FSH-P1 behind the shoulder in beef cows increased to 1.0 or 1.2 ng/mL at 12 h and were back near baseline in approximately 60 h, while FSH levels following im administration of 200 mg NIH-FSH-P1 in 0.5% hyaluronan into Holstein donors reached 1.5 ng/mL at 12 h and returned to baseline in approximately 36 h.

This paper reviews that state of our knowledge concerning follicular wave dynamics, monitoring and manipulation. All camelids have overlapping follicular waves in absence of ovulation which is induced by a seminal plasma factor (βNGF). The interval between follicular waves varies. The size of the ovulatory follicle varies between 11 and 25 mm in camels and between in 6 and 13 mm in South American Camelids. The interval between induction of ovulation and next ovulatory follicle is 15 ± 1 day for all camelids. Follicular activity is best monitored by transrectal ultrasonography. Progesterone therapy for 7–15 days seems to suppress follicular dominance but does not completely inhibit follicular recruitment. Combination of estradiol and progesterone seems to provide better control of follicular activity. Both methods have provided variable results in the synchronization of follicular waves. Combination of induction of ovulation with GnRH and luteolysis at predetermined times shows some promise in synchronization of follicular dominance. These synchronization protocols require further investigation in order to provide practical approaches for fixed-time breeding. Ovarian superstimulation with FSH and eCG alone or in combination is somewhat successful. The best results are obtained when treatment is initiated at the emergence of a new follicular wave after induction of ovulation or following treatment with progesterone for 7–14 days. However, response remains extremely variable particularly in terms of ovulation rate and number of recovered embryos. Sources of this variability need to be studied including the effects of season, nutrition, doses and frequency of administration of gonadotropin.

Made available in DSpace on 2018-12-11T17:18:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-01-15 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The objectives of this study were 1) to monitor corpus luteum (CL) dynamics after two different protocols of ovulation induction in prepubertal Nellore heifers, and 2) to determine differences in luteal function. Fifty-seven heifers (weight 289.61 ± 32.28 kg, BCS 5.66 ± 0.65, age 17.47 ± 0.81 months) were divided into two groups: GP4+GnRH received a progesterone (P4) device of 3rd use for 10 days, followed by the administration of 0.02 mg buserelin acetate (GnRH) 48 h after removal of the device, and GGnRH received only GnRH. The CLs formed were monitored by ultrasonography every 2 days until their functional regression (decrease in the color Doppler signal and serum P4 concentration < 1 ng/mL), determining their diameter and area, numerical pixel value (NPV), pixel heterogeneity, and vascularization percentage. The peak systolic velocity, end diastolic velocity, resistivity index and pulsatility index (PI) of the ovarian artery and serum P4 concentration were also measured. A lifespan of the CL of more than 16 days was classified as normal-function and of less than 16 days as premature regression. The variables were compared between treatments, CL categories (normal-functional, prematurely regressed or non-functional), days of evaluation, and their interactions using the MIXED procedure of the SAS program (p ≤ 0.05). Three animals of each group (6/57 = 11%) did not respond to treatment, corresponding to an ovulation rate of 89%. There was a higher percentage of normal-function CLs in GP4+GnRH (81%) and a higher percentage of non-functional CLs in GGnRH (52%; P4 concentration < 1 ng/mL in all assessments). Normal-function CLs exhibited a greater area, vascularization percentage and P4 concentration than prematurely regressed and non-functional CLs. Lower diameter, area, NPV and P4 concentration were observed for non-functional CLs, but there was no difference in vascularization percentage compared to prematurely regressed CLs. Progesterone concentration was efficient in diagnosing CL function and was positively correlated with CL area (r = 0.62; p < 0.001) and vascularization percentage (r = 0.38; p < 0.001). Diameter and PI were important for the early diagnosis of non-functional and prematurely regressed CLs, respectively. In conclusion, luteal function differed for the first CL that develops after ovulation induction in prepubertal heifers. Ultrasonographic parameters (diameter, area, NPV, vascularization percentage, and PI) can be used to predict CL function. Departamento de Medicina Veterinária Preventiva e Reprodução Animal Universidade Estadual Paulista “Júlio de Mesquita Filho” (FCAV/UNESP) Instituto de Medicina Veterinária Universidade Federal do Pará Instituto de Zootecnia Centro APTA Bovinos de Corte Laboratório de Endocrinologia Animal Faculdade de Medicina Veterinária de Araçatuba (FMVA/UNESP) Departamento de Medicina Veterinária Preventiva e Reprodução Animal Universidade Estadual Paulista “Júlio de Mesquita Filho” (FCAV/UNESP) Laboratório de Endocrinologia Animal Faculdade de Medicina Veterinária de Araçatuba (FMVA/UNESP) FAPESP: 2015/13079-0

Boar exposure is used to stimulate follicle development and estrus in sows after weaning and also to improve semen uptake and sperm transport with insemination. However, the need and value of boar exposure is uncertain when ovulation induction is used. These studies were designed to determine the effect of daily boar exposure after weaning when used with ovulation induction and fixed time post-cervical artificial insemination (PCAI). In experiment 1, sows were weaned into stalls and assigned to receive 3 min of daily fenceline boar exposure (BE, n = 7) or no boar exposure (NBE, n = 8). All sows received OvuGel at 96 h after weaning and BE or NBE 30 min prior to a single PCAI 24 h after OvuGel. Ovaries were assessed daily for follicle size from weaning until ovulation. Cervical contractions were measured 30 min following BE or NBE and before PCAI, while uterine contractions were measured for 1 h following PCAI. In experiment 2, weaned sows (n = 244) were assigned by parity to receive once daily BE for 1.5 min each day or NBE. OvuGel, PCAI and ultrasound methods were performed similarly as in experiment 1. Results from experiment 1 indicated BE did not significantly influence follicle size or measures of fertility. However, BE did increase the frequency of cervical contractions (P 24 h from ovulation but not when inseminations occurred ≤24 h before ovulation. The results indicate that boar exposure for only minutes each day after weaning had beneficial effects for improving follicle development, ovulation induction, and AI timing, most notably in parity 1 sows, but had no beneficial or detrimental effects on the ability to perform PCAI.

This study aimed to investigate the effects of a single administration of one of three different gonadotropins on Day 7 post-insemination on ovarian activity, progesterone (P4) concentration and pregnancy outcomes of rabbit does. Multiparous, non-lactating, V-line does were artificially inseminated after synchronization and ovulation induction with equine chorionic gonadotropin (eCG; 25 IU im) and gonadotropin releasing hormone (GnRH; 0.8 μg buserelin im) 48 h later. On Day 7 post-inseminarion, does were randomly allocated into four groups (n = 40/group). Does of each group were intramuscularly injected with a single dose of one of physiological saline (placebo; control), GnRH (0.8 μg buserelin), human chorionic gonadotropin (hCG; 25 IU) or eCG (25 IU). Concentration of serum P4 was determined on Days 6, 9, 11 and 18 post-insemination. On Day 14 post-insemination, the ovaries and reproductive tracts of pregnant does were removed and weighed. Also, numbers of visible follicles, hemorrhagic follicles, corpora lutea of pregnancy (pCLs), new CLs (nCLs; formed after Day 7 post-insemination) and implantation sites were recorded. Conception rate, parturition rate, abortion rate, litter size/weight and litter viability were recorded. The highest (P < 0.05) reproductive tract and ovary weights were for eCG. The highest (P < 0.05) number of visible ovarian follicles was for eCG, whereas the lowest (P < 0.05) was for GnRH. Treatment with eCG increased (P < 0.05) numbers of pCLs and total implantation sites compared to the other groups. Treatment with GnRH or hCG increased (P < 0.05) number of nCLs compared to control and eCG. The highest rate of fetal loss was in does treated with GnRH. The concentration of serum P4 decreased (P < 0.05) following the treatment with GnRH and continued low until Day 18. However, it remained in line for control, hCG and eCG groups up to Day 11, then decreased (P < 0.05) for control and hCG on Day 18, being lower for hCG than control, while continued to increase for eCG up to Day 18. Compared to control, treatment with eCG improved (P < 0.05) conception and parturition rates by 24 and 22%; respectively, while GnRH and hCG treatments decreased (P < 0.05) them by 57 and 47.6%; respectively. Litter size and litter weight at birth were improved by eCG, but were adversely affectd by GnRH and hCG. In conclusion, a single administration of eCG 7 Days post-insemination could be recommended for improving pregnancy outcomes in rabbits.

Made available in DSpace on 2018-12-11T17:33:44Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-12-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Persistent mating-induced endometritis (PMIE) results in decreased fertility in horses, thereby causing a significant impact in the horse market. Platelet-rich plasma (PRP), a modulator of the inflammatory response, has been largely used in veterinary medicine. Here, we investigated the effects of PRP on uterine inflammation, conception rate, endometrial polymorphonuclear neutrophil (PMN) migration, and COX-2 protein levels in the endometrial tissue. Thirteen PMIE-susceptible mares were used for artificial insemination (AI). The mares were inseminated with fresh semen in three consecutive cycles in a cross-over study design. The following cycle classifications were used: control cycle, no pharmacological interference; pre-AI, 20 mL of PRP was infused 24 h before AI; and post-AI, 20 mL of PRP was infused four h after AI. Follicular dynamics were monitored daily by transrectal ultrasound. When a follicle larger than 35 mm was detected, ovulation was induced with deslorelin acetate (1 mg, im). AI was performed 24 h after ovulation induction. Intrauterine fluid (FLU) was evaluated by ultrasonography before and 24 h after AI. PMNs in uterine cytology (CYT) and biopsy (HIS) were also observed before and 24 h after AI. Pregnancy was determined within 14 days after ovulation. Number of COX-2 positive cells was evaluated by immunohistochemistry. Both PRP treatments resulted in a decrease of PMNs in the CYT after breeding when compared to controls. FLU did not differ between cycles; however, the conception rates were significantly higher in the PRP mares. Mares positive for endometritis decreased in both treatment groups, and a more intense positive COX-2 labeling was observed in the control group when compared to the two treatment groups. In conclusion, PRP beneficially reduces inflammatory response in PMIE mares independent of when treatments were administered, thus increasing chances of successful pregnancy. Department of Animal Reproduction and Veterinary Radiology São Paulo State University – UNESP Santo Amaro University UNISA Severino Sombra University, Vassouras Equine Reproduction Service Department of Animal Medicine and Surgery Faculty of Veterinary Medicine Autonomous University of Barcelona Department of Animal Reproduction and Veterinary Radiology São Paulo State University – UNESP FAPESP: 2015/00150-8

The objective was to compare three shortened protocols for timed-AI (TAI) on ovarian responses, pregnancy per AI (P/AI) and pregnancy loss after resynchronization of ovulation in multiparous Holstein cows. Cows (n = 370), at one location, were randomly assigned at non-pregnancy diagnosis (approximately 32 d after AI) to one of three ovulation resynchronization protocols. Cows in the OS group received a 5-d Ovsynch [100 μg GnRH on Day 1, 500 μg cloprostenol (PGF) on Days 6 and 7, GnRH on Day 8.5 and TAI on Day 9 (16 h after second GnRH)]. Cows in the OS + P4 (progesterone) group received a 5-d Ovsynch as described for OS group plus an intravaginal device (Cue-Mate), containing 1.56 g of progesterone (P4), between Days 1 and 6. Cows in the J-synch group received a Cue-Mate and 2.5 mg of estradiol benzoate (EB) on Day 0, PGF and Cue-Mate removal on Day 6, another PGF on Day 7, and 100 μg of GnRH on Day 8.5, with TAI on Day 9. Ovarian response and pregnancy status at 32 and 60 d after the resynchronization TAI were determined by transrectal ultrasonography. Blood samples were collected at first PGF treatment and at TAI from a subset of 40 cows per group to determine P4 concentrations. Percentage of cows with CL at initiation of the protocol did not differ (P > 0.05) among resynchronization groups. Plasma P4 concentrations at first PGF treatment were greater (P < 0.01) in cows that received a Cue-Mate (OS + P4 and J-synch) compared to OS cows. Luteal regression was greater (P < 0.01) for J-synch (88.6%) compared with OS (76.9%) and OS + P4 (78.8%). More (P < 0.01) cows in the OS + P4 and J-synch groups had their estrous cycle synchronized and were pregnant at 32 d after TAI (48.7 and 34.7%, 67.8 and 48.0%, and 72.4 and 50.0% for OS, OS + P4 and J-synch, respectively). However, more cows subjected to J-synch remained pregnant at 60 d after TAI and, hence, had fewer (P < 0.05) pregnancy losses (19.2, 18.8 and 5.0% for OS, OS + P4 and J-synch, respectively). In summary, cows resynchronized with either OS + P4 or J-synch had greater P4 concentrations at first PGF treatment and a greater response to treatments compared to cows subjected to OS. Although OS + P4 and J-synch resynchronization protocols resulted in increased P/AI at 32 d, pregnancy losses were significantly reduced in cows subjected to the J-synch protocol.

Introducing males to seasonally anestrous females has been shown to increase LH concentrations and hasten the first ovulation in ewes and goat does. Investigation of this sociosexual phenomenon in mares has not been well studied. The objective of this study was to determine if direct or indirect exposure of anestrous mares to a stallion would result in a similar increase in LH and hasten the first ovulation. In late February, mares were stratified by parity and age and assigned randomly to three treatments (n = 7/treatment): (1) DXP (direct exposure; mares housed adjacent to stallion and allowed direct contact for 2 h for 3 d/wk by placing the stallion into a cage in the center of a tease pen); (2) IXP (indirect exposure; mares housed in an adjacent pen to the stallion but not allowed direct contact); and (3) CON (control; mares housed away from stallion and not allowed direct or indirect exposure). During the first, third, and fifth treatment days, blood was collected hourly for 8 h beginning just before treatment initiation for measurement of LH and prolactin (PRL) concentrations. Hormone concentrations were analyzed as repeated measures and time from the first day of treatment to ovulation was analyzed using one-way ANOVA. Interval from treatment initiation to first ovulation did not differ (P 0.05) among treatments: 36.3 ± 6.7, 42.8 ± 4.2, and 48.3 ± 4.2 d for DXP, IXP, and CON, respectively. When comparing the mares based on social status within treatment and level of direct interaction with the stallion, socially dominant DXP mares ovulated sooner (P 0.05) than socially subordinate DXP mares (23.8 ± 3.2 and 53.0 ± 6.8 d, respectively). Although LH concentrations did not differ (P 0.05) among treatments, a significant (P 0.05) day effect was detected with concentrations being greater on the fifth treatment day compared with the first and third days. Concentrations of PRL were greater (P 0.05) in IXP compared with DXP and CON, regardless of treatment day. Regardless of treatment, PRL concentrations were greater (P 0.05) on the third treatment day, compared with the first and fifth days. We conclude that exposure of anestrous mares to a stallion, whether direct or indirect, to anestrous mares failed to elicit the same effects that have been observed in short-day breeders. The effect of stallion exposure on the onset of cyclicity in anestrous mares may be dependent on the duration of direct contact.

The effectiveness of different treatments with recombinant equine FSH to stimulate follicular growth, multiple ovulations and embryo production in seasonally anovulatory mares was evaluated. During mid-winter season (July–August in Argentina, South America) forty light breed donor mares, presenting follicles 35 mm). Ovulations were detected in 80% of mares in Groups 1 and 2, 50% of mares in Group 3 and in none of Group 4 (Control). Among ovulating mares, no differences in number of ovulations, number of embryos recovered, or pregnancy rates were observed among reFSH treatments. Of treated mares, 6, 7, and 5 produced embryos in Groups 1, 2, and 3, respectively. The average embryo recovery rate per ovulated mare was 88%. The average embryo recovery rate per ovulation was 43%. Overall, a 59% pregnancy rate was achieved. These results indicate that treatment with reFSH during deep anestrus results in follicular development, ovulation of fertile oocytes, and production of embryos that established viable pregnancies after transfer. Also, a single daily administration of reFSH was as effective as two daily administrations, which allows for a simplified administration regimen.

Despite postulated potential for wildlife conservation, success of assisted reproduction techniques (ART) in ex-situ feline breeding remains25%. The aim of this project was to develop a simplified, non-surgical artificial insemination (AI) protocol for African lions (Panthera leo), using an exogenous GnRH analogue to induce ovulation in females presenting natural oestrus, and minimizing manipulation of the animals. Four protocols were tested in five trained lionesses (3.5-8 years), for a total of 14 inseminations (2-4 per lioness). These protocols differed in the time lapse between GnRH injection and insemination, on days 4, 5, or 6 from onset of natural oestrus, determined by daily behavioural observation and vaginal cytology. Semen was collected from 8 different males by urethral catheterization and electro-ejaculation, during full anaesthesia. Females were immediately immobilized for AI after semen collection. After transrectal ultrasound examination of the reproductive tract, insemination was performed either intravaginal or transcervical using a commercial dog urinary catheter (2.0 × 500 mm, Buster

Made available in DSpace on 2019-10-06T17:13:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-10-15 Non-steroidal anti-inflammatory drugs (NSAIDs) are a therapeutic option for the treatment of inflammation. However, negative effects of non-selective NSAIDs for treatment of mares with endometritis have been described, including delayed uterine clearance and impairment of ovulations. Firocoxib is a specific cyclooxygenase-2 (COX-2) inhibitor and has the ability to act in the uterus of mares. We investigated the effects of firocoxib on ovulation rate, numbers of polymorphonuclear neutrophils (PMNs), and COX-2 protein levels in the endometrial tissue of susceptible mares after insemination. Two experiments were conducted. In experiment 1, twenty mares were evaluated in two consecutive estrous cycles broken into the following groups: Control - no pharmacological interference; Treatment - mares were treated with 0.2 mg/kg of firocoxib orally. The treatment began on the day of ovulation induction, and firocoxib was administered until one day after artificial insemination (AI). Ovulation was induced with 1 mg of deslorelin acetate and the mares were inseminated 24 h after the injection. Ovulation was confirmed 48 h after induction, and embryos were collected eight days after ovulation. Experiment 2: Nine mares susceptible to persistent mating-induced endometritis (PMIE) were artificially inseminated. The mares were examined with ultrasound and inseminated with fresh semen in two consecutive cycles, control and treated, in a cross-over study design. The amount of intrauterine fluid was measured, and endometrial samples were collected 24 h after AI. The number of PMNs was determined by endometrial cytology and biopsy, and COX-2 labeling in endometrial samples was evaluated by immunohistochemistry. Firocoxib treatment did not induce ovulatory failure or affect embryo recovery rate in Experiment 1. In Experiment 2, firocoxib treatment reduced inflammation after AI in mares as evidenced with results regarding PMN numbers/percentage and endometrial COX-2 staining. In conclusion, the proposed treatment with firocoxib reduced endometrial inflammation in mares susceptible to PMIE after breeding, with no adverse effects. Department of Animal Reproduction and Veterinary Radiology São Paulo State University UNESP Equine Reproduction Service Department of Animal Medicine and Surgery Faculty of Veterinary Medicine Autonomous University of Barcelona LUB Breeding Farm Cesário Lange Department of Animal Reproduction and Veterinary Radiology São Paulo State University UNESP

A reproductive analysis of a captive group of jaguars (Panthera onca; n = 6) at the Santacruz Zoological Foundation in Cundinamarca, Colombia, was conducted by performing a longitudinal, noninvasive, hormonal analysis of estradiol and progestogens in females and of androgens in males. During four seasons, female jaguars confined in solitary were evaluated for ovarian activity and spontaneous ovulation, male jaguars for testicular activity. A second hormonal follow-up was conducted in the females after administration of gonadotropins. Hormones were extracted from fecal samples of three females (n = 3) and two males (n = 2). Estradiol measurements were obtained by RIA and progestogens by enzyme immunoassay. The linear mixed-effect regression showed that there was a significant effect of seasons in the concentrations of estradiol (chi square = 15.97, degrees of freedom = 3, P

Hormonal ovarian superstimulation has contributed to small ruminant reproduction around the world, impacting genetic improvement and zoosanitary programs, contributing to the conservation of endangered species, and supporting other related biotechnologies. Advanced knowledge surrounding the superovulatory treatments in sheep has resulted in enhanced control of influencing factors and improved the protocols currently used. However, in spite of minimization of some adverse factors, superovulatory responses in ewes still remain variable, preventing the more widespread use of superovulation in commercial embryo transfer programs and reproductive research in this species. Recent evidence demonstrates that changes in antral follicular populations and blood supply, and circulating concentrations of certain reproductive hormones determined at the specific time points just before or during the superovulatory treatment are associated with superovulation success in ewes. This review attempts to compile the data from available literature to identify ovarian and hormonal determinants of the superovulatory outcome in ewes, which can be used to substantially improve the existing protocols and to reduce the extra cost and unnecessary stress imposed on poorly responding animals. An overview of most commonly used and some recently developed, FSH-based ovarian stimulation protocols is given at the outset to highlight variation in the frequency and timing of gonadotropin injections, estrus synchronization methods, and follicular wave synchronization and/or ovulation induction techniques during the superovulatory treatments in ewes.

The objective was to study the effects of a commercial pyrethroid-based pour-on product, permethrin, on reproductive performance in superovulated beef heifers by assessing steroid biosynthesis and embryo quality. Nonpregnant, yearling beef heifers (n = 10; 418 ± 33 kg; 5.5 ± 0.2 body conditioning scores) were assigned by body weight and breed to either (1) saline control or (2) permethrin pour-on administered at label dose (PYR). Superovulation was achieved on all heifers using a timed, 17-day, CIDR-based protocol with GnRH and PGF2α and decreasing total dosage of 240-mg FSH administered twice daily for 4 days. Heifers were artificially inseminated twice (at onset of estrus and 12 hours later) by same technician with frozen semen from single bull collection. To determine short- and long-term effects of permethrin on embryo quality and steroid biosynthesis, superovulation was initiated twice with collection of embryos occurring at 17 and 51 days after treatment. Embryos were recovered 6.5 days after first artificial insemination via nonsurgical flush and were evaluated by International Embryo Transfer Society standards. Blood was collected at standing estrus and day of embryo recovery. Estradiol (E2) and progesterone (P4) concentrations were analyzed via RIA. MIXED and GLIMMIX procedures of SAS were used to analyze continuous and categorical data, respectively. Heifer per flush was the experimental unit. Total embryos recovered did not differ because of treatment (P = 0.30), but did decrease in flush 2 compared with flush 1 (P = 0.02). Quality grade, total transferable quality embryos, and overall flush success did not differ because of treatment (P ≥ 0.16). However, transferable quality embryos were decreased in flush 2 compared with flush 1 (P = 0.05). Total unfertilized oocytes were greater in saline control (P = 0.04). The PYR heifers tended to have less total P4 (P = 0.15) and P4 per CL (P = 0.06) at recovery. E2 per ovulated follicle and E2 per total ovarian structure was greater in flush 2 (P ≤ 0.03) but did not differ because of treatment (P ≥ 0.23). In summary, these data indicate that permethrin administration at label dose in superovulated beef heifers has a tendency to reduce P4, but embryo quality is not affected.

This study evaluated the effect of different doses of eCG (control, 300 or 400 IU) administered at progesterone (P4) device removal in suckled Bos taurus beef cows undergoing a timed artificial insemination (TAI) protocol. A total of 966 cows received a P4 insert and 2.0 mg intramuscular estradiol benzoate at the onset of the synchronization. After 9 days, P4 insert was removed, and 12.5 mg of dinoprost tromethamine and 1 mg of estradiol cypionate were administered, followed by TAI 48 hours later. Then, the cows received one of three treatments as follows: control (n = 323), 300 (n = 326), or 400 IU of eCG (n = 317). A subset (n = 435) of cows in anestrus had their ovaries evaluated using ultrasound at the time of P4 removal and at TAI. Data were analyzed by orthogonal contrasts (C): C1 (eCG effect) and C2 (eCG dose effect). Estrous occurrence (control = 53.7%, 300 IU = 70.6%, and 400 IU = 77.0%) and pregnancy per artificial insemination (control = 29.7%, 300 IU = 44.8%, and 400 IU = 47.6%) were improved by eCG treatment (C1; P = 0.0004 and P0.0001, respectively). Furthermore, the cows receiving eCG presented larger follicles at TAI (control = 13.5 ± 0.3 mm, 300 IU = 14.0 ± 0.2 mm, and 400 IU = 15.1 ± 0.3 mm; P0.0001; C1). However, there was no effect of eCG dose on any response variables studied (C2; P0.15). In conclusion, the eCG treatment administered at the time of P4 removal increased the occurrence of estrus, the larger follicles at TAI, and pregnancy per artificial insemination of suckled B taurus beef cows. Despite the greater occurrence of estrus in noncyclic cows receiving 400 IU of eCG, both eCG doses (300 and 400 IU) were equally efficient to improve pregnancy to artificial insemination.

Primiparous and multiparous suckled Bos indicus × Bos taurus cows were used to compare a Select Synch + controlled internal drug release (CIDR) and timed artificial insemination (AI) protocol (SSC) to a modified CO-Synch + CIDR protocol (COS). Five separate groups of cows were utilized (n = 659) and within a group, cows were allotted to two treatments on the basis of body condition score (BCS) taken 10 days before and days postpartum at CIDR insertion (Day 0). Blood samples were collected on Day -10 and 0 for assessment of circulating concentrations of progesterone to determine estrous cycling status. Cows in both treatments received intramuscular (im) GnRH (100 μg) and a CIDR inserted on Day 0. On Day 7, the SSC treatment had CIDR removed and received im PGF2α (25 mg), whereas COS treatment had CIDR removed and received im PGF2α (25 mg) on Day 7.5. In the SSC treatment, estrus was detected daily at 0700 and 1700 hours for 72 hours after PGF2α administration, and cows were artificially inseminated 8 to 12 hours after a detected estrus. Cows failing to exhibit estrus were subjected to timed-AI and received im GnRH (100 μg) 76 ± 4 hours after PGF2α administration. In the COS treatment, cows received im GnRH (100 μg) and were fixed-time artificially inseminated at 64 ± 4 hours after PGF2α administration. Although group differences (P0.05) existed for synchronized AI pregnancy rates, there were no significant interactions of group with any variable analyzed; hence, groups were pooled for further evaluation. Within the SSC treatment, the 72-hour estrous response was 50.6% (168 of 332). Conception rate of cows in estrus was 66.1% (111 of 168), and timed-AI pregnancy rate for nonresponders was 32.3% (53 of 164). Overall synchronized AI pregnancy rates were similar (P0.05) for SSC (49.4%; n = 164 of 332) and COS (47.1%; n = 154 of 327). Estrous cycling status and days postpartum did not (P0.05) influence synchronized AI pregnancy rates but BCS did (P0.05). Cows with a BCS greater than or equal to 5 had synchronized AI pregnancy rates of 50% compared to 39% for cows with BCS lesser than 5.0. In summary, the modified COS protocol yielded similar synchronized AI pregnancy rates compared to the SSC protocol, and it appears that the modified COS protocol has the potential to be an effective timed-AI protocol in suckled Bos indicus × Bos taurus cows.

Control of ovarian function in cheetahs is sub-optimal, which currently limits the integration of assisted reproductive techniques into the genetic management of that endangered species. The objective of this study was to determine the effect of preemptive progestin treatment on the quality of ovarian responses after exogenous gonadotropin stimulation in cheetahs. Adult females received either 1) 200 IU equine chorionic gonadotropin (eCG) followed with 3,000 IU porcine luteinizing hormone (pLH) (intramuscular route) (n = 5; control group) or 2) similar eCG/pLH administration preceded by a 7-day treatment with oral progestin (0.1 mg/kg altrenogest; ALT group; n = 7). At 42 h post-pLH administration, a series of metrics was assessed via laparoscopy (number of follicles ≥ 2 mm, number of corpora lutea, oviduct and uterine cornua diameter and overall vascularization). Concentrations of fecal estradiol, progesterone and glucocorticoid metabolites (FEM, FPM, and FGM, respectively) were measured by enzyme immunoassay for 3 wk before ALT treatment (Period 1), 7 d during ovarian suppression period (Period 2), throughout eCG/LH treatment and laparoscopy (Period 3), and 6 wk following laparoscopy (Period 4). Overall, nine out of 12 cheetahs (4/5 in control and 5/7 ALT group) had freshly-formed corpora lutea at the time of laparoscopy. Mean follicle and corpora lutea numbers in the control versus ALT group were not different (P > 0.05). Overall measurements and vascularization scores also did not differ (P > 0.05) among groups. FEM average concentrations increased (P ≤ 0.05) in response to eCG for the ALT-treated females between Periods 2 and 3 and were sustained during Period 4. However, FEM average concentrations did not vary (P > 0.05) for control females throughout Periods 1–4. Post-ovulatory FPM average concentrations (Period 4) did not differ (P > 0.05) between the ALT-treated females and controls. FPM average concentration from both groups increased in Period 4 compared to Periods 1–3 (P ≤ 0.05). Females receiving the ALT treatment also had lower (P ≤ 0.05) FGM metabolite average concentrations than control females during ovarian suppression (suggesting adrenal suppression). Collective results suggest that ovarian response to gonadotropin treatment in the cheetah was improved following oral progestin administration due to the normative increase in estradiol following stimulation for these females compared with control. This treatment should lead to more effective timed assisted reproduction procedures for this species.

The objective of this study was to determine the estrous response and the moment of ovulation and fertility after a progesterone (P4) priming plus human chorionic gonadotropin (hCG) administration in multiparous and nulliparous goats. Therefore, two experiments were conducted during non-breeding season (April and May, 26° N) and all the animals received a single injection of 20 mg of P4 and 100 IU of hCG 24 h later. In Experiment 1, 13 multiparous and 9 nulliparous goats were subjected to estrus detection twice a day from P4 administration, and their ovaries were scanned by transrectal ultrasonography every 12 h from the onset of estrus to determine ovulation. The proportion of goats in estrus did not differ between multiparous and nulliparous females. The onset of estrus (60.5 ± 12.4 h vs. 52.0 ± 5.2 h after hCG administration) and the moment of ovulation (91.5 ± 10.3 h vs. 85.3 ± 11.5 h) were also similar in multiparous and nulliparous goats. In Experiment 2, a total of 299 multiparous and nulliparous goats managed under intensive (n = 112 and 41 goats, respectively) or extensive (n = 85 and 61 goats, respectively) production systems were divided to receive a fixed-time artificial insemination (FTAI) with fresh semen at 60 or 72 h after hCG administration. The pregnancy rate did not differ significantly between multiparous and nulliparous goats in both production systems. Nonetheless, in the intensive system pregnancy rate was affected by the moment of insemination (P 0.05). In this system, the pregnancy rate was higher in goats inseminated at 60 h than in 72 h (55.6%, 44/79 vs. 35.1%, 26/74, respectively; P 0.05). On the contrary, in the extensive system the pregnancy rate was not affected by the time of insemination (29.4%, 23/78 vs. 22.0%, 15/68). To conclude, both the ovulatory response and the pregnancy rate after a single P4 injection plus hCG was similar between multiparous and nulliparous goats during anovulatory season. Although the pregnancy rate was not affected by the time of insemination in the extensive production system, under intensive conditions FTAI should be performed at 60 h after hCG treatment.

This study was conducted to develop simple superovulation protocols in dromedary camels. Using two commercial FSH products, a series of experiments was conducted to evaluate the effect of different superovulation protocols on ovarian response and embryo production. In experiment 1, camels in control group (n = 15) received 400 mg Folltropin-V in a traditional protocol (FSH diluted in saline and given twice daily in decreasing doses over 5 days) and camels in split-injection (2 doses 48 h apart) groups received either 400 (n = 16, first dose: 320 mg, second dose: 80 mg) or 200 mg (n = 16, first dose: 120 mg, second dose: 80 mg) of slow-release (SR) preparation of Folltropin-V [Folltropin-V diluted in hyaluronan (5 mg/mL) solution]. In experiment 2, camels in control group (n = 13) received 2000 IU Pluset in a traditional protocol and camels in split-injection groups received either 2000 (n = 14, first dose: 1600 IU, second dose: 400 IU) or 1000 IU (n = 16, first dose: 600 IU, second dose: 400 IU) of SR preparation of Pluset (Pluset diluted in hyaluronan solution). In experiment 3, camels received SR preparation of 200 mg Folltropin-V (n = 45, first dose: 120 mg, second dose: 80 mg) or 1000 IU Pluset (n = 42, first dose: 600 IU, second dose: 400 IU) in a split-injection protocol. In experiments 1 and 2, the mean number of ovulations, corpora lutea, transferable embryos and unfertilized ova were similar (P > 0.05) between groups. In experiment 3, there was no difference (P > 0.05) between SR preparations of Folltropin and Pluset on the number of ovulatory sized (≥9 mm) follicles (15.8 ± 0.9 vs 16.7 ± 0.9) and transferable embryos (5.0 ± 0.4 vs 5.2 ± 0.5). In conclusion, split-injection of SR preparation of FSH resulted in a similar superovulatory response compared to a traditional protocol and a similar superovulatory response can be achieved with SR preparations of Folltropin-V and Pluset in a split-injection protocol.

The aim of this study was to evaluate the pattern of expression of LHCGR isoforms in Gir heifers characterized as good (10.3 ± 1.2 ova/embryos per flush, n = 5) or poor responders (1.1 ± 0.3 ova/embryos per flush, n = 5) to superovulation protocols. In both groups, an adapted ultrasound-guided follicular aspiration system was used to collect granulosa cells from 8 mm follicles formed either during a synchronized, non-stimulated follicular wave (no stimulation control, NS) or on the fourth day of a superovulation protocol (SOV) induced with 200 IU of pFSH. The recovered follicular fluid was centrifuged and granulosa cells were washed with NaCl 0.9% and kept in RNAlater®. RNA extraction was performed using a commercial RNeasy Micro Kit and eluted samples were quantified and reverse transcribed using the commercial Superscript III kit. cDNA samples were amplified by real-time PCR using a primer to target LH/hCG receptor gene - not selective for LHCGR isoforms (total LHCGR) - and four sets of isoforms selective primers (S1, S10, S10 + 11, and S11). Analyses were performed using the REST software and expression levels are shown as mean ± SEM. Under physiological conditions (NS), poor responders had a higher expression of total LHCGR (4.9 ± 1.7 fold-change, P

The use of strategies to stimulate follicular growth are important, especially for use in timed artificial insemination (TAI) protocols, aiming to increase dairy cow's fertility. The aim of this study was to investigate the effect of insulin on follicular growth, steroid production and expression of genes related to follicular development. For this, cows were submitted to a progesterone (P4) and estradiol (E2) based synchronization protocol. In study 1, eleven primiparous lactating Holstein cows, received a single s.c. application of 0.25 IU/kg human insulin or no treatment (control) on D8 of the protocol. Blood samples were collected, and the dominant follicle diameter was assessed daily via transrectal ultrasonography, from D8 to D12. In study 2, eight multiparous non-pregnant and non-lactating Jersey cows, received a single s.c. application of 0.25 IU/kg human insulin, whereas cows from the control group received a single s.c. injection (1 mL) of saline solution (NaCl 0.9%). Blood samples were collected, and the dominant follicle diameter was assessed daily via transrectal ultrasonography from D6 to D9 of the protocol. Sixteen hours after insulin injection, follicular aspiration was performed. In study 1, insulin treatment decreased systemic glucose levels, but did not affect follicular growth. In study 2, the glucose decrease induced by insulin treatment was accompanied by a tendency of decreased progesterone levels in follicular fluid, along with a decrease in steroidogenic acute regulatory protein (STAR) and insulin like growth factor binding protein 2 (IGFBP2) mRNA abundance in granulosa cells. In conclusion, insulin treatment does not increase follicle growth and estradiol secretion in dairy cows, but decreases IGFBP2 and tends to increase pappalysin (PAPPA) mRNA abundance in granulosa cells, suggesting a positive effect on follicle development.