Successful Vitrification of In vivo Embryos Collected from Superovulated Japanese Black Cattle (Wagyu)

The aim of this study was to determine whether vitrification is an effective method when used for Japanese Black Cattle (Wagyu) in vivo‐derived embryos, collected following a superovulation treatment and embryo transfer (MOET) programme. In vivo‐derived morula and blastocysts collected on day 7 after artificial insemination, were vitrified using a modified droplet vitrification (MDV) procedure and subsequently warmed for transfer (ET) into synchronized recipients. Fresh embryos, and embryos cryopreserved using a standardized slow freezing procedure (direct thaw/direct transfer, DT) served as ET controls. Two different follicle‐stimulating hormone (FSH) sources, Folltropin^® Canada (FSH BAH, 24 donors) and a brand prepared by the Chinese Academy of Science (FSH CAS, 16 donors), were compared in a series of superovulation outcomes following well‐established FSH administration protocols. Following data analysis, the total number of ovulations recorded at the time of embryo flushing (10.5 vs 8.5; p = 0.28) and the total number of transferable embryos (6.2 vs 5.1; p = 0.52) were similar between the two FSH sources. ET for MDV (39.7%, n = 78), DT (35.2%, n = 71) and fresh controls (47.1%, n = 34) resulted in similar pregnancy rates (p > 0.05). When MDV was used, a higher pregnancy rate (42.6%) resulted from the transfer of vitrified morulae, when compared to the DT counterparts (24.3%), (p = 0.05). Transfer of vitrified morulae resulted also in higher pregnancy rate, when compared to the transfer of vitrified blastocysts (42.6% vs. 29.4%; p < 0.05). Transfer of DT blastocysts resulted in higher pregnancy rate than morulae, similarly cryopreserved (47.1% vs. 24.3%, p < 0.05). In conclusion, MDV is an effective alternative methodology for cryopreservation of in vivo‐derived embryos. This study gives also indication that, compared to vitrified blastocysts, MDV of morula stage embryos results in higher pregnancy rates following warming and transfer into synchronized recipients.     

Multiple Factors Affecting Superovulation in Poll Dorset in China

To expand the breeding flock of Poll Dorset sheep in China, multiple ovulation and embryo transfer breeding program was applied to the limited number of imported Australian Poll Dorset sheep. This study investigated the effects of FSH from three different manufacturers, parity (nulliparous vs multiparous), repeated superovulation, oestrus induction, corpus luteum regression and oestrus delay on Poll Dorset superovulation. The results showed that gonadotropin FSH from Canada Folltropin‐V (Ca‐FSH) was successfully used for superovulatory treatment with 160 mg–200 mg dosage per ewe and recovered 12.91 ± 7.80 embryos. Multiparous ewes for superovulation treatment were significantly better nulliparous ewes (p < 0.05). The successive superovalution treatment reduced significantly embryo collection but did not affect transferable embryo number. Ewes with natural oestrus resulted in significantly higher number of embryos (13.83 ± 4.64) and of transferable embryos (12.00 ± 5.76) than ewes with induced oestrus (7.00 ± 4.92; 4.22 ± 3.42) and unknown oestrus cycle (5.94 ± 3.38; 3.19 ± 2.56, p < 0.05). The delayed oestrus ewes at 24 h after superovulatory treatment produced significantly fewer embryos and transferable embryos (0.92 ± 1.51 vs 0.42 ± 0.90) than those with normal oestrus (p < 0.01). Furthermore, the more transferable embryos were recovered from ewes with normal corpus luteum than those with corpus luteum regression (5.88 ± 5.09 vs 3.59 ± 4.30 and 8.83 ± 5.75 vs. 6.66 ± 5.41, p < 0.01). These results suggest that in our farm practice, a comprehensive treatment method by using the Canadian FSH (Folltropin‐V), plus choosing multiparous and natural oestrus ewes with normal corpus luteum might obtain an optimum embryo collection and embryos transfer in sheep.     

Ovulatory Response and Embryo Yield in Jakhrana Goats Following Treatments with PMSG and FSH

Superovulatory response and embryo production efficacy were investigated in adult (age 2–4 years, average body weight: 27–43 kg) cycling Jakhrana goats (n = 15) under semi-arid environmental conditions of India by administering different superovulatory regimens. Goats were reared under semi-intensive system of management in established farm conditions. To synchronize oestrus, a luteolytic dose of carboprost tromethamine (Upjohn, UK) was administered intramuscularly to all does at the dose rate of 5μg per kg body weight in a double dose schedule with an interval of 11 days. For superovulation, 750 IU of PMSG (Folligon, Intervet, Boxmeer, Holland) per goat was administered intramuscularly 24 h before administering a second dose of luteolytic agent in five does (treatment 1). FSH (Sigma, St. Louis, MO, USA) 12.50 IU per goat was administered intramuscularly in a decreasing daily dose schedule (2.50, 2.50; 1.875, 1.875; 1.25, 1.25; 0.625, 0.625) at 12 h intervals over four days, initiated 48 h before administering second dose of carboprost tromethamine in 5 does (treatment 2). FSH (Super-Ov, Ausa Intern, USA) was administered at a uniform dose rate of 8.33 units per goat intramuscularly at 24 h intervals over three consecutive days (total dose was 25 units), initiated 48 h before administering a second dose of carboprost tromethamine in 5 does (treatment 3). To synchronize ovulation in responders, human chorionic gonadotrophin (hCG, Chorulon, Intervet) was injected intramuscularly at a dose rate of 500 IU in each goat on the day of oestrus appearance. Goats were laparotomized 72–82 h following the onset of synchronized oestrus and their genitalia were flushed using a standard collection procedure. Variability (p > 0.05) in superovulatory response (number of established corpora lutea) was observed: FSH (Sigma), 11.8± 2.9; FSH (Super-Ov), 11.6±4.5; PMSG (Intervet), 8.4±2.3. A similar pattern was reflected in mean embryo and transferable embryo recovery, respectively (p > 0.05): FSH (Sigma), 8.0±1.8, 5.2±1.7; FSH (Super-Ov), 6.6±2.4, 5.4±2.4; PMSG, 5.8±1.9, 3.8±2.2. In PMSG-treated does, comparatively more unfertilized ova or retarded embryos were recovered than in FSH-treated does. The superiority of FSH preparations over PMSG was reflected in terms of total and transferable embryo production (p > 0.05). On average, five transferable embryos (excellent and good quality) were recovered per doe treated with FSH of either source. The mean ova/embryo recovery was satisfactory (55–68%). Results indicated that Jakhrana goats can be superovulated for embryo production using FSH of either source to augment productivity.     

Pregnancy rate after fixed‐time transfer of cryopreserved embryos collected by non‐surgical route in Lacaune sheep

This study investigated the feasibility of applying fixed‐time (cryopreserved) embryo transfer in ewes. Embryos (n = 106) were non‐surgically recovered from superovulated donors (n = 39) on day 6–7 after oestrus. Straws containing one or two embryos (morulae and/or blastocysts) subjected to either slow freezing (SF, n = 62) or vitrification (VT, n = 44) were randomly used within fixed‐time embryo transfer on Day 8.5. Recipient ewes were nulliparous (n = 58) bearing corpora lutea after synchronous oestrous induction protocol. The pregnancy rate was higher (p = .03) in SF (39.4%) than VT (16.9%) and survival rate tended (p = .08) to be higher in SF than in VT (25.8% vs. 15.9%). Lambing rates were similar (p = .13) between SF (20.9%) and VT (15.9%). Embryos recovered by non‐surgical route after cervical dilation treatment and later cryopreserved by either slow freezing or vitrification produced reasonable pregnancy rates after FTET.     

Superovulatory response and embryo quality in Katahdin ewes treated with FSH or FSH plus eCG during non-breeding season

The aim of this study was to evaluate the effect of a co-treatment of follicle-stimulating hormone (FSH) plus equine chorionic gonadotrophin (eCG) on serum insulin and insulin-like growth factor 1 (IGF-1) concentrations, superovulatory response, ovulatory rate, and number and production of embryos in Katahdin breed ewes during the non-breeding season. Twenty Katahdin ewes were synchronized with progestagens (CIDR) and assigned to two superovulation treatments (n = 10): (1): ewes treated with 200 mg ewe⁻¹ of FSH from day 5 to 8 after CIDR insertion at decreasing doses every 12 h (FSH group) and (2) ewes treated as FSH group plus 300 IU of eCG on day 5 after CIDR insertion (FSH + eCG group). Estrous behavior was monitored and direct mating was performed. On days − 7 (CIDR insertion), 0 (CIDR withdrawal), and 7 (embryo recovery), blood samples were collected to determine serum hormone concentrations. Co-treatment with eCG (FSH group) did not affect (P > 0.05) serum hormone levels. Superovulation response, ovulation rate, recovery rate, fertilization, and number of embryos were also similar (P > 0.05) between treatments. Compared with FSH group, FSH + eCG ewes had lower (P < 0.05) number of transferable embryos and higher (P < 0.05) number of oocyte and a tendency to increase the number of degenerated embryos (P = 0.07). Overall results suggest that the administration of eCG is not beneficial either to improve the ovulatory response or the amount of transferable embryos in Katahdin ewes superovulated with a protocol using progesterone and FSH at decreasing doses.

     

Superovulation protocols for dairy cows bred with SexedULTRA™ sex‐sorted semen

The objective was to compare embryo yield and quality in lactating dairy cows superovulated (SO) with varying amounts of gonadotropins and FSH:LH ratios and inseminated with SexedULTRA? sex‐sorted semen. The SO treatments (n = 77) involved 3 protocols: groups F700 and F1000 were given total doses of 700 and 1,000 IU of Folltropin (FSH:LH ratio 49:1), respectively, whereas group F700P300 was given 700 IU of Folltropin + 300 IU of Pluset (FSH:LH ratio 1:1). Cows were artificially inseminated 3 times over a 10‐hr interval with frozen‐thawed SexedULTRA? sex‐sorted semen (total of 10 × 10⁶ sex‐sorted sperm), starting 18 hr after onset of oestrus, with embryos/ova recovered 7 d after oestrus. Total number of recovered structures and transferable embryos were lower (p < 0.05) in F700 (4.7 ± 3.0 and 1.9 ± 1.7, respectively; mean ± SD) compared to F1000 (8.1 ± 3.8 and 4.4 ± 2.6) and F700P300 (8.5 ± 6.4 and 4.5 ± 3.3). Percentage of cows ovulating >50% of follicles ≥0.8 cm in diameter was lower (p < 0.05) in F700 (35.5%) than in F1000 (82.4%) and F700P300 (73.1%). Percentage of unfertilized oocytes was higher (p < 0.05) in F700 (45.0% vs. 27.7% for F1000 and 29.0% for F700P300) whereas percentage of morulae was higher (p < 0.05) in F1000 (19.3% vs. 8.7% for F700 and 12.2% for F700P300). Embryo quality was similar among groups (p > 0.05). In conclusion, embryo production in lactating dairy cows was improved by increasing total dose of gonadotropins from 700 to 1,000 IU, with SexedULTRA? sex‐sorted semen yielding satisfactory fertilization rates and embryo quality.     

Differential gene expression of Eph‐ephrin A1 and LEPR‐LEP with high or low number of embryos in pigs during implantation

The objective of this study was to ascertain whether mRNA and protein expressions of implantation‐related genes (erythropoietin‐producing hepatocellular receptor–ligand A1, Eph‐ephrin A1 and leptin receptor–leptin, LEPR‐LEP) differed between pigs with high and low number of embryos, and whether these differences in gene expression might affect embryo implantation. Experimental pig groups (n = 24) for high and low number of embryos were prepared by altering the number of eggs ovulated in pre‐pubertal gilts treated with 1.5 × (High) or 1.0 × (Low) PG600 ([400 IU PMSG + 200 IU hCG]/dose, AKZO‐NOBEL). Gilts expressing oestrus were artificially inseminated twice and maintained in breeding and gestation until the reproductive tract was collected on day 22 of pregnancy. At slaughter, the reproductive tracts from each pregnant gilt from each treatment were immediately processed to collect samples for RNA and protein analysis. Within each gilt, three conceptus points were sampled, one from each horn and then a random conceptus within the tract. At each conceptus point, endometrial attachment site, chorion–allantois and embryo were collected and immediately frozen in liquid nitrogen. Number of corpus luteum (CL) (35.4 vs. 12.6) and total embryo number (18.8 vs. 10.2) were greater in the high‐embryo compared to the low‐embryo group, respectively (p < .05). Real‐time qPCR results showed that Eph‐ephrin A1 mRNA expression was less in the high‐embryo (p < .05) compared to the low‐embryo group. In addition, Western blotting analysis indicated that Eph‐ephrin A1 and LEP protein expression at endometrial attachment site in high‐embryo was less (p < .05) compared to low‐embryo group. It was also noted that mRNA expression of Eph‐ephrin A1 and LEPR‐LEP was greater in pregnant than non‐pregnant gilts (p < .05). Moreover, mRNA expression of Eph‐ephrin A1 (p < .05) and LEPR‐LEP was greatest at endometrial attachment site among all three tissues. There was a positive correlation between expressions of Eph‐ephrin A1, LEPR‐LEP and embryo length with the correlation coefficient 0.31–0.59. For Eph‐ephrin A1, the highest correlation coefficient appeared between Eph A1 expression and normal embryo number, between ephrin A1 expression and embryo length. For LEPR‐LEP, the highest correlation coefficient appeared between LEPR‐LEP expression and ovary weight (0.79 for both, p < .05), followed by embryo length and weight. The results of this study suggest that low expression of Eph‐ephrin A1 and LEPR‐LEP is somehow related to increased embryo number during implantation and that endometrial attachment site might be the main target tissue of these gene products. Yet, the increased expression of Eph‐ephrin A1 and LEPR‐LEP appeared associated with increased embryo growth (length and weight) and ovary weight, Eph‐ephrin A1 and LEPR‐LEP might play roles in the regulation of embryo implantation in pigs.     

Is the Production of Embryos in Small‐Scale Farming an Economically Feasible Enterprise?

The present assay attempts to evaluate the feasibility of using embryo transfer in small community farmers by in vivo study and by modelling the results obtained. From the total of 59 donor cows, 62.7% responded to treatment, with a significant difference (p = 0.002) in the percentage of the response between breeds, being 90.5% (19/21) in Holstein and 47.4% (18/38) in Brahman. A total of 283 embryos were graded as transferable, while 141 as non‐transferable, without difference in the percentage of transferable embryo by breed (p = 0.18). The mean of transferable embryos graded as class I and II was not different between Holstein and Brahman (p = 0.96 and p = 0.92, respectively); besides, no differences were observed in the other grades (non‐transferable). The highest difference in costs, regardless of its quality by breed, was seen in the lower levels of probable fertility of the embryo transferred, even reaching several hundred dollars. When modelling the expected costs for embryo produced and transferred, values can reach nearly $2000.00 when the probable fertility is only 10%. However, when the probable fertility was 60%, embryo cost was close to $300.00. This technology seems to be viable on average or high‐scale systems, having a superovulatory response between 60 and 80% with 4–6 transferrable embryos. Yet, in small‐scale farming, due to the reduced number of donors and/or recipients, the costs surpass the economical feasibility of the technique.     

Elevated cyclin D1 expression is governed by plasma IGF‐1 through Ras/Raf/MEK/ERK pathway in rumen epithelium of goats supplying a high metabolizable energy diet

The objective of this study was to explore the underlying mechanism of insulin‐like growth factor 1 (IGF‐1)–caused cell proliferation of rumen epithelium in goats fed a high metabolizable energy (ME) diet. In this study, young goats were fed either a low ME [LL, n = 9, ME: 0.57 MJ/kg^0.75/day] or high ME [HL, n = 9, ME: 1.00 MJ/(kg^0.75/day)] diet for 42 day. The time duration of G₁‐phase was shortened as a result of enhanced expression of cyclin D1 mRNA in the HL group (p < 0.05). It was suggested that a high ME diet promoted cell transition from G₀/G₁ to S‐phase via cyclin D1. The level of phosphorylation of ERK was higher in HL than LL group (p < 0.05). In cell culture, the ERK was phosphorylated by IGF‐1 treatment. The proliferative effects of insulin‐like growth factor 1 (IGF‐1, 25 ng/ml) on [³H] thymidine (TdR) incorporation into DNA and on cyclin D1 protein expression of rumen epithelial cells were inhibited by PPP (the inhibitor of type 1 IGF receptor) (p < 0.05) and ERK inhibitor (p < 0.05) in vitro. Thus, IGF‐1 up‐regulated cyclin D1 expression and accelerated G₁‐phase progression in the cell cycle through Ras/Raf/MEK/ERK pathway in rumen epithelium of goats.     

Effect of hCG and eCG Treatments on Prostaglandins Synthesis in the Porcine Oviduct

The oviduct plays a crucial role in fertilization, gamete maturation and embryo transport. Prostaglandins are some of the main factors determining its roles. The present study investigated the influence of oestrus synchronization and superovulation on prostaglandins synthesis in the porcine oviduct. Mature cross‐bred gilts after exhibiting oestrous cycles were divided into four groups: I, cyclic; II, inseminated; III, synchronized and inseminated; and IV, superovulated and inseminated. Oviducts were collected on the third day of the oestrous cycle or after insemination and divided into isthmus and ampullary parts. This study demonstrated lower mRNA (in the isthmus and ampulla; p < 0.05, p < 0.001, respectively) and protein prostaglandin endoperoxide synthase 2 expression (in the isthmus; p < 0.001) in gilts treated with human chorionic gonadotrophin/equine chorionic gonadotrophin (hCG/eCG) compared with Group II that were inseminated only. In addition, hCG and eCG treatment decreased mPGES‐1 mRNA levels in Groups III and IV, in both the isthmus (p < 0.01 in III, p < 0.001 in IV) and ampulla (p < 0.001). The prostaglandin E₂ content of oviductal tissues was significantly lower in Groups III (p < 0.05) and IV (p < 0.01 in isthmus, p < 0.0001 in ampulla) compared with Group II. mRNA and protein levels of PGFS in Group IV in the oviductal isthmus were higher (p < 0.01) compared with the non‐treated Group II. In effect, the amount of prostaglandin F_2α in oviductal tissues of gilts treated with hCG/eCG was significantly elevated (p < 0.001 in isthmus of Groups III and IV; p < 0.05 in ampulla of Group IV). Differential expression of the factors analysed in gilts treated with exogenous gonadotrophins suggests that hCG/eCG stimulation affects prostaglandins synthesis pathway. These changes can alter oviduct functions and in turn affect gamete maturation and fertilization as well as development of embryos and their transport to the uterus.     

La récolte et le transfert d'embryons chez la chèvre Angora

The objective of this study was to analyze the efficiency of embryo collection and transfer in Angora goats during the breeding season. Synchronization was obtained by placing an intravaginal sponge for either a short period of time (11 days + PGF_2α) or a long period (17 to 21 days). Thirty-three goats were superovulated, embryo collection was not attempted on seven goats (four did not respond to the superovulation treatment, three did not show estrus), and four gave oocytes only. The embryos of one goat are still frozen. The remaining 21 goats produced 285 embryos of which 187 (66%) were transferred (fresh or thawed) to 66 recipients; 36 (55%) recipients remained pregnant and gave birth to 63 kids (34% of the transferred embryos). The superovulation and transfer techniques used in this study provided a large number of transferable embryos. However, the percentage of embryos that reach term could be improved by more strict selection of the recipients and of the embryos to be transferred, and by improving the embryo freezing technique.     

Secondary Corpora Lutea Induced by hCG Treatment Enhanced Demi‐Embryo Survival in Lactating High‐Yielding Dairy Cows

Using a novel in vivo model considering a low developmental competence embryo (demi‐embryo) and a subnormal fertility recipient (lactating high‐yielding dairy cow), this experiment evaluated the effect of human chorionic gonadotrophin (hCG) treatment at embryo transfer (ET) on embryonic size at implantation, embryonic survival and recipient plasma progesterone (P₄) and bovine pregnancy‐specific protein B (PSPB) concentrations until day 63 of pregnancy. Embryos were bisected and each pair of demi‐embryos was bilaterally transferred to recipients (n = 61) on day 7 of the oestrous cycle. At ET recipients were randomly assigned to treatment with 1500 IU hCG or to untreated controls. Higher (p < 0.01) pregnancy rates on days 25, 42 and 63, and embryo survival rate on day 63 were observed in hCG‐treated cows with secondary CL than in hCG‐treated cows without secondary CL and in untreated cows. Pregnancy rates and embryo survival rate were similar in hCG‐treated cows without secondary CL and untreated cows. Embryonic size on day 42 was not affected by treatment with hCG, presence of secondary CL and type of pregnancy (single vs twin). Presence of secondary CL increased (p < 0.05) plasma P₄ concentrations of pregnant cows on days 14, 19 and 25 but not thereafter and of non‐pregnant cows on days 14–21. Treatment with hCG and presence of secondary CL had no effect on plasma PSPB concentrations, which were higher (p < 0.05) in twin than in single pregnancies. In conclusion, secondary CL induced by hCG treatment at ET significantly increased plasma P₄ concentrations, the survival rate of demi‐embryos and the pregnancy rate of high‐yielding lactating dairy cows. Embryos were rescued beyond maternal recognition of pregnancy, but later embryonic survival, growth until implantation and placental PSPB secretion until day 63 of pregnancy were not affected by treatment or presence of secondary CL.     

Effect of Oestrus Synchronization with PGF2α/eCG/hCG on Luteal P4 Synthesis in Early Pregnant Gilts

Administration of hormones to synchronize oestrus is a useful tool in animal breeding. However, exogenous ovarian stimulation may be detrimental to reproductive function. This study was aimed to examine whether an oestrus synchronization with PGF2α/eCG/hCG could affect luteal P4 synthesis in early pregnant gilts. Corpora lutea (CLs) were collected on days 9, 12 and 16 of pregnancy from gilts with natural (n = 16) and synchronized (n = 18) oestrus and analysed for (i) the expre‐ssion of steroidogenic acute regulatory protein (StAR), cytochrome P450 family 11 subfamily A polypeptide (CYP11A1), and 3β‐hydroxysteroid dehydrogenase (3βHSD); (ii) the concentration of P4 in the luteal tissue and blood; and (iii) the expression of luteinizing hormone receptors (LHR) and oestrogen receptors (ERα and ERβ). Additionally, the effect of LH on P4 secretion from CL slices collected from synchronized and naturally ovulated animals has been studied in vitro. PGF_2α/eCG/hCG administration increased mRNA expression of StAR, CYP11A1, 3βHSD, and LHR on day 9 and CYP11A1 and LHR on day 12 of pregnancy compared with the control group (p < 0.05). CYP11A1, 3βHSD, LHR, ERα and ERβ proteins were not affected by synchronization; only StAR protein increased in hormonally treated animals (p = 0.017). The concentration of P4 in luteal tissue was greater on day 9 (p < 0.01), but lower on day 16 (p < 0.05) in gilts with hormonally induced oestrus compared with control animals. Blood serum levels of P4 were lower in synchronized than control gilts (p < 0.001). Synchronization did not affect LH‐stimulated P4 secretion from luteal slices; however, greater basal concentration of P4 in incubation medium was detected for CLs collected from synchronized than control gilts (p < 0.05). In conclusion, synchronization of oestrus with PGF2α/eCG/hCG protocol in gilts did not impair the expression of luteal P4 synthesis system, although decreased P4 concentration in the blood.     

Antral Follicle Populations and Embryo Production – In Vitro and In Vivo – of Bos indicus–taurus Donors from Weaning to Yearling Ages

Interest in indicus–taurus cattle has been increasing, as these animals are likely to present the best characteristics of Zebu and European bovine breeds. The aim of this study was to compare the embryo production of indicus–taurus donors with high vs low antral follicle counts obtained by ovum pickup/in vitro production (OPU/IVP) and superovulation (SOV)/embryo collection. Braford females at weaning age (3/8 Nelore × 5/8 Hereford, n = 137, 9 ± 1 month old) were subjected to six serial ovarian ultrasonographs and were assigned to two groups according to the number of antral follicles ≥3 mm as follows: G‐High antral follicular count (AFC, n = 20, mean ≥40 follicles) and G‐Low AFC (n = 20, mean ≤10 follicles). When the females (n = 40) reached 24 months of age, they were subjected to both OPU/IVP and SOV/embryo collection. The average number of follicles remained highly stable throughout all of the ultrasound evaluations (range 0.90–0.92). The mean number of COCs recovered (36.90 ± 13.68 vs 5.80 ± 3.40) was higher (p < 0.05) for females with high AFC, resulting in higher (p < 0.05) numbers of total embryos among females with high vs low AFC (6.10 ± 4.51 vs 0.55 ± 0.83). The mean number of embryos per collection was also higher (p < 0.05) for G‐High vs G‐Low (6.95 ± 5.34 vs 1.9 ± 2.13). We conclude that a single ultrasound performed at pre‐pubertal ages to count antral follicles can be used as a predictor of embryo production following IVP and SOV/embryo collection in indicus–taurus females.     

Ovarian and Hormonal Responses to Follicular Phase Administration of Investigational Metastin/Kisspeptin Analog,TAK‐683, in Goats

This study evaluated the effects of follicular phase administration of TAK‐683, an investigational metastin/kisspeptin analog, on follicular growth, ovulation, luteal function and reproductive hormones in goats. After confirmation of ovulation by transrectal ultrasonography (Day 0), PGF_2α (2 mg/head of dinoprost) was administered intramuscularly on Day 10 to induce luteal regression. At 12 h after PGF_2α administration, intravenous administration of vehicle or 35 nmol (50 μg)/head of TAK‐683 was performed in control (n = 4) and treatment (n = 4) groups, respectively. Blood samples were collected at 6‐h intervals for 96 h and then daily until the detection of subsequent ovulation (second ovulation). After the second ovulation, ultrasound examinations and blood sampling were performed every other day or daily until the subsequent ovulation (third ovulation). Mean concentrations of LH and FSH in the treatment group were significantly higher 6 h after TAK‐683 treatment than those in the control group (12.0 ± 10.7 vs 1.0 ± 0.7 ng/ml for LH, 47.5 ± 28.2 vs 15.1 ± 3.4 ng/ml for FSH, p < 0.05), whereas mean concentrations of oestradiol in the treatment group decreased immediately after treatment (p < 0.05) as compared with the control group. Ovulation tended to be delayed (n = 2) or occurred early (n = 1) in the treatment group as compared with the control group. For the second ovulation, ovulatory follicles in the treatment group were significantly smaller in maximal diameter than in the control group (3.8 ± 0.5 vs 5.4 ± 0.2 mm, p < 0.05, n = 3). Administration of TAK‐683 in the follicular phase stimulates gonadotropin secretion and may have resulted in ovulation of premature follicles in goats.     

Efficiency of Oestrous Synchronization by GnRH,Prostaglandins and Socio‐Sexual Cues in the North African Maure Goats

This study aims to develop at different seasons, for local North African Maure goats, synchronizing protocols simultaneously to the standard ‘S’ protocol using progestagens in association with prostaglandins and gonadotropin. In late May, 40 goats were assigned to either the ‘S’ protocol or to a protocol where oestrus and ovulation were induced by the buck effect in single‐injection progesterone‐treated goats and provoking early luteolysis using prostaglandin 9 days after exposure to bucks ‘B’. During the 72 h after the treatments ended, 15 and 5 goats expressed oestrus in the ‘S’ and ‘B’ protocols (p < 0.01). Mean time to oestrus was shorter for ‘S’ than for ‘B’ goats. Ovulation rate averaged 2.1 ± 0.22 and 1.60 ± 0.35 for, respectively, ‘S’ and ‘B’ goats (p > 0.05). During mid‐September, 60 goats were assigned to either ‘S’ treatment, ‘PGF’ treatment where oestrus and ovulation were synchronized using two injections of prostaglandin 11 days apart or to ‘GnRH’ treatment where the goats had their oestrus and ovulation synchronized with a GnRH (day 0)–prostaglandin (day 6)–GnRH (day 9) sequence. More ‘S’ goats were detected in oestrus over the 96‐h period after the end of the treatments (88.8, 73.7 and 55% in ‘S’, ‘PGF’ and ‘GnRH’ treatments, respectively; p < 0.05). Mean ovulation rates were 2.3 ± 0.27, 1.33 ± 0.27 and 1.33 ± 0.27 for, respectively, ‘S’, ‘PGF’ and ‘GnRH’ goats (p < 0.001). Despite a similar ovulatory response to ‘S’ protocol, efficiency of prostaglandin and GnRH‐based treatments should be tested in mid‐breeding season.     

Embryo Production in Superovulated Goats Treated with Insulin Before or After Mating or By Continuous Propylene Glycol Supplementation

Seventeen adult and cyclic Moxoto goats were synchronized using 60 mg MPA vaginal sponge for 11 days and 50 μg cloprostenol, 48 h before sponge removal, and superovulated with 120 mg pFSH i.m. in decreasing doses at 12 h intervals for three consecutive days. In seven goats, 0.2 IU/kg BW/day of long acting insulin was subcutaneously injected at same time as pFSH, and in the other five goats, the same dose of insulin was injected for three consecutive days starting 24 h after mating. Finally, five goats were supplemented with an oral dose of 80 ml/goat/day of propylene glycol continuously during the experiment. The animals were flushed at 7 days after mating and the embryos were classified based on International Embryo Transfer Society criteria. Blood samples were collected every 3 days for insulin assay. Administration of insulin raised the insulin levels of the goats (p < 0.05), whereas in the group treated with propylene glycol, insulin rate was different only between FSH treatment and after mating (p < 0.05). Similar rates of recovery for total (80.05 ± 9.78%) or transferable structures (61.03 ± 15.13%) were obtained. Treatment was not influenced (p > 0.05) by responsiveness to superovulation, which averaged 64%. By contrast, insulin treatments were shown to increase the number of embryos considered excellent with respect to goats supplemented with propylene glycol (p < 0.05). When insulin was given before mating, a strong relationship (r = 0. 90) (p < 0.05) between number of transferable embryo and ovulations was observed in the animals. In conclusion, superovulated goats treated with low doses of exogenous insulin resulted in an enhancement in embryo quality, which was related to changes in circulating insulin concentrations.     

Determination of the relationship between serum anti‐Müllerian hormone level and superovulatory response in Simmental cows

The most significant focal points of the embryo transfer technology are as follows: the selection of donors, the response of the selected donor to the superovulation protocol and the obtained number of the transferable embryos. For this purpose, it is suggested that donor selection can be done by anti‐Müllerian hormone (AMH) levels, and embryo production is evaluated. AMH is secreted by the granulosa cells of primordial, pre‐antral and antral follicles below 4 mm in the ovary, independent of FSH. Therefore, the aim of this study was to investigate the relationship between serum AMH levels and the number of corpus luteum (CL), total embryos and transferable embryos that were shaped after a uniform superovulation protocol. For this reason, 48 Simmental cows, which were located at General Directory of Agricultural Enterprises (region, province, etc. instead of the general directorate), were used as donors for the embryo transfer. Blood samples were taken at random, regardless of the stage of animal's sexual cycle. AMH levels were measured by enzyme‐linked fluorescent assay (ELFA) method of the miniVIDAS^® (bioMérieux SA) using AMH Bovine Test Kit. According to the statistical analyses of the obtained data, AMH levels were positively correlated with CL and total embryos (p < .05). No significant correlations between AMH and transferable embryos were approved (p > .05). It was also determined that each 200 pg/ml increase in serum AMH level resulted in one increase in CL number. Overall, considering the positive correlation between AMH level and the obtained number of CL and total embryos after a superovulation treatment, it was concluded that measuring blood AMH level prior to any further costly implementation may be an effective method in donor selection.     

Effect of season and superstimulatory treatment on in vivo and in vitro embryo production in wood bison (Bison bison athabascae)

Two experiments were done using a two-by-two design to determine the effects of season and superstimulatory protocol on embryo production in wood bison. In Experiment 1 (in vivo-derived embryos), ovarian superstimulation was induced in female bison during the ovulatory and anovulatory seasons with either two or three doses of FSH given every-other-day (FSH × 2 vs. FSH × 3, respectively). Bison were given hCG to induce ovulation, inseminated 12 and 24 hr after hCG, and embryos were collected 8 days after hCG (n = 10 bison/group). In Experiment 2 (in vitro embryo production), ovarian superstimulation was induced in female bison during the ovulatory and anovulatory seasons with two doses of FSH, and in vivo maturation of the cumulus–oocyte complexes (COC) was induced with hCG at either 48 or 72 hr after the last dose of FSH. COC were collected 34 hr after hCG, and expanded COC were used for in vitro fertilization and culture. In Experiment 1, the number of follicles ≥9 mm, the proportion of follicles that ovulated, the number of CL, and the total number of ova/embryos collected did not differ between seasons or treatment groups, but the number of transferable embryos was greater (p < .05) in the ovulatory season. In Experiment 2, no differences were detected between seasons or treatment groups for any end point. The number of transferable embryos produced per bison was greatest (p < .05) using in vitro fertilization and was unaffected by season (1.5 ± 0.2 and 1.1 ± 0.3 during anovulatory and ovulatory seasons, respectively), in contrast to in vivo embryo production which was affected by season (0.1 ± 0.01 and 0.7 ± 0.2 during anovulatory and ovulatory seasons, respectively). Results demonstrate that transferable embryos can be produced throughout the year in wood bison by both in vivo and in vitro techniques, but the efficiency of embryo production of in vivo-derived embryos is significantly lower during the anovulatory season.     

Effects of pistachio by‐products on digestibility,milk production,milk fatty acid profile and blood metabolites in Saanen dairy goats

The objective of this study was to investigate the effects of pistachio by‐products (PBP) on nutrient digestibility, blood metabolites and milk fatty acid (FA) profile in Saanen dairy goats. Nine multiparous lactating Saanen goats (on day 90 post‐partum, 45 ± 2/kg BW) were randomly assigned to a 3 × 3 Latin square design with three treatment diets: 1) control diet (alfalfa hay based), 2) 32% PBP and 3) 32% PBP + polyethylene glycol (PEG‐4000; 1 g/kg dry matter). Each period lasted 21 days, including 14 day for treatment adaptation and 7 day for data collection. Pistachio by‐products significantly decreased (p < 0.01) crude protein (CP) digestibility compared with the control diet (64.4% vs. 58.7%), but PEG addition did not differ for CP digestibility of goats fed 32% PBP + PEG and those fed the two other diets. The digestibility of NDF tended (p = 0.06) to decrease for goats fed PBP compared with those fed the control diet. Yields of milk and 4% fat‐corrected milk were not affected by dietary treatments. Compared with the control diet, PBP supplementation appreciably changed the proportions of almost all the milk FA measured; the main effects were decreases (p < 0.01) in FA from 8:0 to 16:0 and increases (p < 0.01) proportions of cis‐9, trans‐11 18:2 and trans‐11 18:1, monounsaturated FA, polyunsaturated FA and long‐chain FA. The saturated FA, short‐chain FA and medium‐chain FA proportions were lower (p < 0.01) in goats fed the two PBP supplemented diet than in those fed the control diet and PEG addition led to intermediate proportions of saturated FA, unsaturated and monounsaturated FA. Inclusion of PBP in the diet decreased (p < 0.01) plasma concentrations of glucose and urea nitrogen compared with the control diet. It was concluded that PBP can be used as forage in the diet of dairy goats without interfering with milk yield. Inclusion of 32% PBP in the diet of dairy goats had beneficial effects on milk FA profile but PEG addition to PBP did not contribute to enhance further milk FA profile.