Effects of different extenders, storage temperatures, and antioxidant supplementation on chilled semen quality: a review.

The successful preservation of ram semen is essential to promote genetic variability, ensure semen transportation, and inseminate multiple ewes. Currently, either animal or plant-based lipoprotein-based extenders are used for semen preservation. Animal product-based extenders include milk and egg yolk, while soybean lecithin is a plant-based extender. Although extenders containing products of animal origin better preserve the quality of chilled semen, the in vivo efficacy after 24 h of storage is still of great concern. Storage temperature is another important and effective factor in preserving sperm quality, whereby different storage temperatures are adopted to enhance the storage life of sperm. Low temperatures (4-5 °C) better preserve sperm quality for a longer duration than high temperatures (15, 20, and 25 °C). Moreover, antioxidant supplementation has a positive impact on sperm quality during liquid storage. The current review summarizes the outcomes of various extenders, different storage temperatures, and antioxidant supplementation on the liquid storage of ram sperm.

The presence of estradiol benzoate in the cervical relaxation treatment for non-surgical embryo collection does not impair embryonic morphological quality, cryosurvival, and gene expression profile.

Non-surgical embryo recovery (NSER) is usually preceded by a cervical relaxation in ovine donors, based on estradiol benzoate (EB), prostaglandin (PGF), and oxytocin (OT). However, it is hypothesized that, due to poorly understood mechanisms, EB can result in embryotoxic actions. To evaluate this, 20 min before NSER superovulated sheep were induced to cervical relaxation with 0.0 (G0.0), 0.5 (G0.5), or 1.0 mg (G1.0) of EB associated with 37.5 µg of PGF 16 h before NSER and 50 IU of OT. In doing so, the efficiency and duration of the NSER procedure showed no compromise (P > 0.05). Additionally, the presence of EB did not affect (P > 0.05) the embryo's morphological quality, the development dynamics, or the abundance of transcripts associated with embryonic quality (OCT4 and NANOG), cellular stress (HSP90 and PRDX1), and apoptosis (BCL2 and BAX). A similar result (P > 0.05) was also observed when comparing embryonic cryosurvival at 24 (52.0, 52.0, and 54.0) and 48 h (60.0, 54.0, and 58.0) of in vitro culture (G0.0, G0.5, and G1.0, respectively). Thus, we can conclude that EB use does not compromise embryonic quality and cryoresistance.

Intravaginal progesterone device reinsertion during the early luteal phase affects luteal function and embryo yield in superovulated ewes.

This study checked the efficacy of progesterone (P4) device reinsertion during the early luteal phase on luteal function and embryo yield in superovulated crossbred ewes. Twenty multiparous ewes received an intravaginal P4 device for nine days (D0 to D9) followed by six decreasing doses (25, 25, 15, 15, 10, 10%) of 133 mg pFSH i.m. at 12 h intervals, starting 60 h before P4 device removal. Ewes were naturally mated at 12 h intervals while in estrus. On D13, ewes with viable corpora lutea (CL; n = 19) were equally allocated for receiving their P4 device reinsertion (G-P4; n = 10) or not (G-Control; n = 9). On D17, the P4 device was removed, and all females received the cervical relaxation protocol 16 h to 20 min before non-surgical embryo recovery. CL count and their functionality classification were performed on D13 and D17 by transrectal B-mode and color Doppler ultrasonography (US). Plasma P4 concentrations (ng/mL) of G-P4 ewes increased (P < 0.05) over the days, being greater on D17 (9.2 ± 2.8) than on D9 (1.9 ± 0.7) and D13 (1.6 ± 0.4). The overall CL count per ewe tended to be greater (P = 0.09) in G-P4 compared with G-Control. The occurrence of premature regression of corpora lutea did not differ (P > 0.05) between G-P4 (30.0%) and G-Control (44.4%). The number of ova/embryos recovered was greater (P < 0.05) in G-P4 (11.6 ± 2.9) compared with G-Control (3.7 ± 2.0), respectively. Altogether, the reinsertion of the P4 device for four days after superovulation in ewes promotes greater P4 concentrations, resulting in greater ova/embryos recovered.

Role of phosphoinositide 3-kinase/ protein kinase B/ phosphatase and tensin homologue (PI3K/AKT/PTEN) pathway inhibitors during in vitro maturation of mammalian oocytes on in vitro embryo production: A systematic review.

Modulation of phosphoinositide 3-kinase/protein kinase B/phosphatase and tensin homologue (PI3K/AKT/PTEN) pathway in mammals yields mixed results. A deep understanding of its regulation can be a powerful tool for better in vitro blastocyst production. This systematic review aims to map the evidence of PI3K/AKT/PTEN pathway modulation during in vitro maturation (IVM), to assess its effects on meiosis resumption and nuclear maturation progression of mammalian oocytes, and their impacts on embryo development and quality. A total of 1058 articles were screened in three databases, and 22 articles were included. Fifty-two IVM assessments were identified, among which 11 evaluated blastocyst yield. Three PI3K inhibitors (3-methyladenine, Wortmannin, and LY294002) and one AKT inhibitor (SH6) were investigated. The impact of this pathway modulation on meiosis resumption in swines and murines was not well established, depending on the inhibitor used, concentration, and media supplementation, while in bovines, resumption seems to be independent of PI3K/AKT/PTEN pathway. However, progression to metaphase II (MII) is highly controlled by this pathway on both bovines and swines. Studies that focused on the inhibition reversibility showed that the removal of the modulator produced MII rates similar to the control group. Experiments that aimed to temporarily block meiosis resumption or reduce PI3K activity resulted in blastocyst production equal to or even higher than control groups. Altogether, these data indicate the paramount potential of this pathway as a possible strategy to improve overall in vitro embryo production efficiency, by synchronizing both nuclear and cytoplasmic maturation.

Progestogen supplementation during superovulation leads to higher embryo viability and TGFB1 gene expression in sheep.

This study aimed to compare the effect of the administration of either medroxyprogesterone acetate (MPA) or progesterone (P4) in superovulation (SOV) treatments applied during the first follicular wave on follicular development, embryo yield, and the expression of genes related to pluripotency maintenance, differentiation of the trophectoderm, cell growth and differentiation, apoptosis and energy metabolism in sheep embryos. The estrous cycle of 36 multiparous ewes was synchronized with a short protocol, and the animals were randomly allocated to three groups. At the beginning of SOV, 12 ewes per treatment received an intravaginal sponge impregnated with 60 mg of MPA (TMPA), or an intravaginal device containing 0.33 g of P4 (TP4), or received no progestogen treatment (CON). The device was kept until the fifth dose of FSH. Ewes were mated with five fertile rams. Gene expression was performed by RT-qPCR using grade I and II blastocysts. The numbers of corpora lutea, total structures and viable embryos recovered per ewe were similar (P > 0.05) among groups. However, the viability rate was higher in TP4 (71.9 ± 16.3%) compared to CON (24.4 ± 16.8%; P = 0.01) and similar to TMPA (49.9 ± 16.3%; P = 0.2). Similarly, when compared with CON, treatment with P4 or MPA positively regulated the TGFB1 transcript involved in cell proliferation and differentiation (P = 0.01 and P = 0.03, respectively). In conclusion, supplementation with P4 during the first follicular wave of the estrous cycle improves embryo viability and alters the expression of the TGFB1 gene.

Hormonal protocol used for cervical dilation in ewes does not affect morphological embryo quality but reduces recovery rate and temporarily alters gene expression.

BACKGROUND: Information on the impact of hormonal protocols for cervical dilation on the quality of ovine embryos is scarce. METHODS: To compare the quality of embryos after cervical dilation protocol, ewes (n = 64) were allocated into either a treated group (100 µg estradiol benzoate intravenous and 0.12 mg cloprostenol intramuscularly, 12 hours before embryo collection plus 100 iu oxytocin intravenous 15 minutes before the collection procedure) or a control group (saline). Luteal function was analysed using ultrasonography and P4 measurement. Some collected embryos were frozen/thawed for gene expression, others were cultured in vitro, frozen/thawed for gene expression, and the remaining embryos were fixed for the apoptosis test (TUNEL test). RESULTS: The treatment reduced fluid (p=0.04) and structure (p=0.03) recovery rates, but the morphological quality, development stage, and apoptosis incidence of the embryos were not affected by treatment. The corpora lutea of the control group had greater blood perfusion (p = 0.002) and greater P4 concentrations at 6, 9, and 12 h after the treatment (p < 0.0001). The expression of BAX, BCL2, PRDX1, and HSP90 genes were not affected by the treatment. However, the embryos in the treated group had fewer NANOG and OCT4 transcripts than control embryos (p = 0.008; p = 0.006, respectively). After culture, there was no difference between the groups in any gene. CONCLUSION: The hormonal protocol for cervical dilation reduced the efficiency of embryo collection. In addition, the treatment induced luteolysis and a transient alteration of embryo gene expression, however there were no detectable changes in embryo morphological quality, development stage, or incidence of apoptosis.

Effect of eCG in a short-term synchronization treatment on ovarian status, estrus synchrony, and ovulation in dairy goats managed under tropical conditions.

The aim of this study was to assess the need of using eCG on short-term estrus synchronization protocol in nulliparous (NUL) and multiparous (MULT) dairy goats during the breeding season. Alpine (n = 20), Nubian (n = 20), and Saanen (n = 16) goats received 60 mg medroxyprogesterone acetate intravaginal sponges for 6 days plus 30 µg d-cloprostenol and 200 IU eCG (G-eCG, n = 28) or saline (G-Control, n = 28) 24 h before sponge removal. The NUL and MULT goats of each breed were equally assigned into the two treatments. Transrectal ultrasonography was used to evaluate ovulatory parameters, and teaser goats were used for estrus detection every 12 h from sponge removal to ovulation. eCG did not affect (P > 0.05) estrus response (~86%), diameter of ovulatory follicles (~6.8 mm), and number of ovulations (~1.6). Nevertheless, eCG led to earlier (P < 0.05) ovulation (G-eCG = 65.1 and G-Control = 73.2 h) and increased (P < 0.05) the ovulation rate (G-eCG = 96.4% and G-Control = 67.9%). In the absence of eCG, no differences regarding reproductive parameters (P > 0.05) were found between parity orders. Alpine MULT goats underwent a superior (P < 0.05) number of ovulations (2.2) in comparison to NUL goats (1.3). In conclusion, the exclusion of eCG from short-term estrus synchronization protocol did not interfere with estrus response but decreased the ovulation rate.

Vaginal cytology and cervical mucus as tools to predict ovulation time in small ruminants.

The possibility of using cervical mucus and vaginal cytology as tools to predict ovulation time was assessed in 11 ewes and 11 does raised under tropical conditions. Every 12 h from progesterone removal to ovulation, estrus behavior, cervical mucus, vaginal cytology, and ovarian ultrasound exams were performed. In goats, vaginal cytology had 88% of accuracy on detecting the ovulation time. However, in sheep, there was no cell pattern in the vaginal cytology and cervical mucus varied at ovulation. In conclusion, both vaginal cytology and mucus evaluation may be useful tools to determine the ovulation time in goats; however, both strategies are less accurate in sheep.

Porcine oocyte preincubation in oviductal fluid flush before in vitro fertilization in the presence of oviductal epithelial cells improves monospermic zygote production.

The present study was designed to evaluate the effect of the combination of oviduct fluid flush (OFF) and oviduct epithelial cells (OEC) in modulating the incidence of polyspermy in pigs. Therefore, for in vitro fertilization (IVF), oocyte and sperm were co-cultured in Tris-buffered medium (TBM) either supplemented with 10% OFF (OFFD group), or in the presence of a bovine OEC monolayer (OEC group), or the oocytes were exposed to OFF for 30 min before IVF (OFFB group), or in the presence of an OEC monolayer (OFFB + OEC group). Regardless of sperm concentration used (0.5, 1.5, and 4.5 × 105 cells/ml), supplementation of IVF medium with 10% OFF led to an increased (P < 0.05) monospermy rate, without alteration (P > 0.05) of the penetration rate in comparison with the control and OEC groups. When the IVF medium was supplemented with heparin, an overall increase (P < 0.05) of the final output of the IVF system in terms of zygotes with two pronuclei (2PN) was observed in the OFFD group, compared with the control and OEC groups, at a sperm concentration of 4.5 × 105 cells/ml. At this concentration, OFFB improved the monospermy rate but decreased the penetration rate, resulting in low efficiency of monospermic zygotes production. Despite this, no major effect was observed in the developmental competence of the presumed zygotes up to the blastocyst stage. The combination of OFFB with OEC improved the penetration rate, while maintaining the high monospermic rate induced by OFFB. In conclusion, the combination of treatment of oocytes by diluted OFF 30 min before IVF, followed by IVF in the presence of OEC, improved monospermic zygote production without reducing the penetration rate, when the IVF medium was supplemented with heparin.

Luteotropic effects of human chorionic gonadotropin administered 7.5 days after synchronous estrous induction in Morada Nova ewes.

This study was conducted in ewes to assess effects of human chorionic gonadotropin (hCG) administration after imposing an estrous induction treatment regimen. Ewes (n = 115) were treated with a 60 mg medroxyprogesterone-intravaginal-sponge for 6 d plus 200 IU of equine chorionic gonadotropin (eCG) im and 37.5 µg d-cloprostenol im 36 h before sponge removal (Day 0). After natural mating, ewes having at least one corpus luteum (CL; n = 108) were administered either 1 mL of saline (G-Control; n = 53) or 300 IU of hCG (G-hCG; n = 55) on Day 7.5 after sponge removal (Day 0). Ovarian ultrasonography and blood collection were performed on Days 7.5, 13.5, 17.5, 21.5, and 30.5. Accessory CL (aCL) were observed in 81.5 % (G-hCG) and 0.0 % (G-Control) of ewes (P = 0.0001). Diameter, area, and volume of luteal tissue were greater (P < 0.05) in G-hCG from Day 13.5 to 30.5. Progesterone (P4) concentrations were greater (P < 0.05) on Days 13.5, 17.5, 21.5 and 30.5 for ewes of the G-hCG group. Pregnancy percentage was similar (P = 0.25) between groups [47.1 % (G-control) compared with 60.0 % (G-hCG)], although total number of lambs produced by estrous synchronized ewes was greater (P = 0.005) in ewes of the G-hCG group (90.9 % compared with 66.0 %). In conclusion, hCG administration 7.5 days after sponge removal from Morada Nova ewes during the non-breeding season is an effective treatment to induce aCL formation, improve luteal tissue biometry and P4 concentrations, and to enhance the total number of lambs born.

Use of two cloprostenol administrations 11.5 days apart efficiently synchronizes oestrus in photostimulated multiparous dairy goats in the non-breeding season.

This study assessed the efficiency of synchronous oestrous induction by light programme followed by two doses of cloprostenol in acyclic Saanen goats of different parity orders. Primiparous (n = 22) and multiparous (n = 33) goats were subjected to 16 hr of light and 8 hr of darkness for 60 days (D0-D60), starting 10 days after the winter solstice. All goats received 120 µg cloprostenol doses on D130 (morning) and D141.5 (afternoon) (11.5 days apart). Oestrus behaviour, ovarian follicular dynamics and serum progesterone (P4) analyses were recorded from D0 to D174 at different intervals. Animals in oestrus after D141.5 were randomly assigned into two groups: assisted natural mating (NM) or artificial insemination (AI; 10-24 hr after oestrus onset with frozen-thawed semen). From D57 to D120, 89.0% of goats presented large follicles (5-8 mm) and P4 concentrations were subluteal from D0 to D120. More multiparous compared to primiparous goats (54.5%, 18/33 vs. 18.2%, 4/22) exhibited oestrus after both injections. More primiparous compared to multiparous goats (54.5%, 12/22 vs. 12.1%, 4/33) did not exhibit oestrus after any injection. A total of 35 goats (64%) were in oestrus after the second prostaglandin injection and were subjected to NM or AI. The conception rate was similar among primiparous (70.0%, 7/10) and multiparous (68.0%, 17/25) goats but the pregnancy rate differed, being 31.8% (7/22) and 51.5% (17/33), respectively. No interaction was found between parity order and P4 concentrations in does that became pregnant or not. Thus, the association between light programme (60 days, starting at the beginning of winter) and two cloprostenol administrations 11.5 days apart (starting 70 days after the end of the light treatment) resulted in sufficient synchronous oestrous response in multiparous acyclic Saanen goats to reach satisfactory fertility levels after both NM and AI.

Transcervical vs. laparotomy embryo collection in ewes: The effectiveness and welfare implications of each technique.

This study assessed animal welfare in ewes subjected to transcervical (TC) or laparotomy (LP) embryo collection, and the efficiency of these two techniques. Santa Inês ewes (n = 57) received a protocol for estrus synchronization and superovulation. Cervical dilation protocol was initiated 12 h before embryo collection in all ewes. Depending on the success of cervical passage, the embryos were collected from ewes by either TC or LP. Records were made of physiological (rectal temperature (RT) and heart rate (HR)), endocrine (cortisol concentration), biochemical (glycaemia, total proteins, globulin and albumin concentrations), and behavioral variables. Data were recorded before fasting (BF) and sedation (BS), during (DC) and immediately after embryo collection (IAC), and 1 h (1hAC), 3 h (3hAC), 6 h (6hAC), 12 h (12hAC), 24 h (24hAC), and 48 h (48hAC) after embryo collection. The LP and TC procedures were applied to 22 and 35 ewes (with 100.0% and 94.3% of procedures being successful, respectively). The use of LP took longer than TC (P = 0.007) but was less effective in the recovery of uterine fluid and structures (P = 0.0002 and P = 0.0180, respectively), with no difference in the number of viable embryos recovered per animal. The TC procedure induced a greater RT at DC (P = 0.002) and IAC moments (P < 0.0001). The heart rate was greater in TC than LP in IAC (P = 0.036). On the other hand, HR was greater with LP at 12hAC (P = 0.033) and 24hAC (P = 0.002). There was no interaction between the procedures and time on total proteins, albumin, or globulin concentrations. The TC procedure induced greater glycaemia than LP in IAC (P < 0.0001). LP induced greater serum cortisol concentration than TC at DC, IAC, 1hAC (P = 0.0004; P = 0.0006; P = 0.036, respectively), even though it was greater in the TC than the LP procedure at 3hAC (P = 0.008). In conclusion, the TC embryo collection was more effective than the traditional LP procedure. Although both embryo collection procedures affected ewes' welfare, the TC procedure is probably less stressor than the LP.

Successful transcervical uterine flushing can be performed without or reduced dose of oestradiol benzoate in cervical relaxation protocol in Dorper ewes.

This study assessed the efficiency of cervical relaxation protocol using none, half or full dose (1.0 mg) of oestradiol benzoate in Dorper ewes subjected to non-surgical embryo recovery (NSER). Thirty-six pluriparous ewes received progestogen sponge (60 mg) for 9 days plus eCG administration (300 IU i.m.) 24 hr before sponge removal. Ewes were not mated and were randomly assigned to receive at 16 hr before NSER 37.5 µg d-cloprostenol i.m. and different doses of oestradiol benzoate: 0.0 mg (0EB group; n = 12); 0.5 mg (0.5EB group; n = 12) or 1.0 mg of oestradiol (1.0EB group, n = 12). All ewes received oxytocin (50 IU) i.v. 20 min before NSER, which was performed 8 days after sponge removal. Corpora lutea were counted by transrectal ultrasonography 24 hr before NSER. After procedure, the ewes were kept in natural breeding period to check their post-NSER fertility. NSER was performed in 91.7% (33/36) of the animals with overall fluid recovery efficiency over 97% (p > .05). The cervical transposing with Hegar dilator was longer (p < .05) in 0EB (4.2 ± 0.3 min) compared to 0.5EB (1.7 ± 0.3 min) and 1.0EB group (1.5 ± 0.3 min). The cervical transposing with mandrel/catheter was longer (p < .05) in 0EB (2.4 ± 0.5 min) than 1.0EB group (1.3 ± 0.5 min). Overall duration of uterine flushing was 25.4 min with structure recovery rate of 43.5%, with no difference among groups (p > .05). The post-NSER fertility was higher (p < .05) in 0.0EB (90%) than 0.5EB group (36.4%). In conclusion, NSER can be successfully performed in Dorper ewes by using a cervical relaxation protocol without oestradiol benzoate.

Efeito da técnica de coleta e do estágio do ciclo estral na recuperação de oócitos de boa qualidade em felinos e caninos domésticos / The effect of recovery technique and stage of estrous cycle on the recoveryof good quality oocytes in domestic felines and canines

A obtenção de oócitos de boa qualidade é essencial para o sucesso de diversas biotécnicas reprodutivas. Objetivou-se determinar o efeito de duas técnicas na recuperação de oócitos de boa qualidade em gatas e cadelas em diferentes estágios reprodutivos. Foram utilizados 43 pares de ovários de gata e 35 de cadela após realização da ovariosalpingohisterectomia eletiva. A fase do ciclo estral foi classificada em inativa, folicular ou luteal. Os ovários da fase folicular foram divididos em três grupos: PUN) punção dos folículos com agulha; PUN+FAT) fatiamento do mesmo ovário já puncionado; e FAT) fatiamento do segundo ovário. Os ovários das fêmeas em fase luteal e inativa foram submetidos ao FAT. Foram obtidos no total 974 oócitos (~23/animal) nas fêmeas felinas e 940 (~27/animal) nas caninas. O fatiamento recuperou número superior (P0,05) entre as técnicas de coleta na qualidade de estruturas recuperadas. A quantidade de oócitos recuperados em cada fase foi similar (P>0,05). Contudo, a fase inativa foi superior à luteal (P<0,05) e semelhante à folicular na quantidade de oócitos de boa qualidade em gatas e não houve diferença em cadelas. Conclui-se que o fatiamento recupera maior quantidade de oócitos, não influenciando em sua qualidade. As fases inativa e folicular recuperam maior quantidade de oócitos de boa qualidade em gatas e não afetam a recuperação em cadelas. Portanto, para otimizar o uso das biotecnologias, deve-se levar em consideração o estágio do ciclo estral em fêmeas felinas e a técnica de coleta utilizada na recuperação de oócitos.

The recovery of good quality oocytes is essential for the success of various reproductive biotechniques. The aim of this study was to determine the effect of two techniques on the recovery of good quality oocytes in queens and bitches at different reproductive stages. A total of 43 pairs of ovaries of queens and 35 of bitches after elective ovariosalpingohisterectomy were performed. The estrous cycle phase was classified as inactive, follicular or luteal. The ovaries of the follicular phase were allocated into three groups: PUN) puncture of the follicles with a needle; PUN + SLI) slicing of the same ovary already punctured; and SLI) slicing of the second ovary. The ovaries of luteal and inactive females were submitted to SLI. A total of 974 oocytes (~23/animal) were obtained in feline females and 940 (~27/animal) in canines females. The SLI technique recovered superior number (P0.05) between the collection techniques in the quality of recovered structures. The number of oocytes recovered in each phase was similar (P>0.05). However, the inactive phase was higher than luteal (P<0.05) and similar to the follicular phase in the quantity of good-quality oocytes in queens and there was no difference in bitches. In conclusion, it is preferable to perform the slicing technique to recover more oocytes in both species. Moreover, in queens it is possible to obtain good quality oocytes in the inactive phase and in bitches the estrous cycle phase does not influence the quality.

Combined treatment with oestradiol benzoate, d-cloprostenol and oxytocin permits cervical dilation and nonsurgical embryo recovery in ewes.

This study examined the feasibility of transcervical embryo recovery after the hormonal treatment to induce cervical dilation, following the 7-day oestrous synchronization protocol in multiparous Santa Inês ewes. A total of 23 cyclic ewes received two doses of 37.5 µg of d-cloprostenol by latero-vulvar route 7 days apart. After the second injection of d-cloprostenol, the ewes were checked for oestrus (every 12 hr) and then mated by fertile rams throughout the oestrous period. All ewes received 37.5 µg of d-cloprostenol (latero-vulvar) and 1 mg of oestradiol benzoate by either intramuscular (EBim group; n = 12) or intravaginal (EBivg group; n = 11) route 16 hr before embryo flushing. Twenty minutes before the flushing, 50 IU of oxytocin were administered intravenously. The oestrous response (i.e., the percentage of ewes that showed signs of oestrous behaviour after the second d-cloprostenol injection) was 91.3% (21/23). The proportion of successfully penetrated ewes (81.8% compared with 80.0%), the mean duration of embryo flushing (24.7 ± 2.0 min compared 26.2 ± 1.9 min), the flushing fluid recovery rate (94.8 ± 1.3% compared with 91.0 ± 2.9%) and the average number of structures recovered per ewe (0.5 ± 0.4 compared with 0.8 ± 0.4) did not vary (p > 0.05) between the EBim and EBivg groups. Viable embryos were recovered from 41.2% (7/17) of successfully penetrated ewes. It can be concluded that nonsurgical (i.e., transcervical) embryo collection can be performed in oestrous-synchronized Santa Inês ewes pretreated with d-cloprostenol, oxytocin and oestradiol benzoate, with the latter hormone administered by either the intramuscular or intravaginal route.

Role of cAMP modulator supplementations during oocyte in vitro maturation in domestic animals.

Cyclic adenosine monophosphate (cAMP) is an important molecule in signal transduction within the cell, functioning as a second cell messenger of gonadotrophin stimulation. The concentration of cAMP in cumulus-oocyte complexes (COCs) is known to be controlled through modulation of its synthesis by adenylyl cyclase (AC) and by degradation through the cyclic nucleotide phosphodiesterase (PDE) enzymes. One of the main obstacles for in vitro embryo production is the optimization of reproduction processes that occur in oocyte maturation. The function of cAMP is important in maintaining meiotic arrest in mammalian oocytes. When the oocyte is physically removed from the antral follicle for in vitro maturation (IVM), intra-oocyte cAMP concentrations decrease and spontaneous meiotic resumption begins, due to the depletion of inhibitory factors from the follicle. In many studies, relatively greater cAMP concentrations before IVM has been reported to improve oocyte competence, leading to subsequent benefits in embryonic development in different species. There, therefore, has been an increase in oocyte cAMP concentrations with several treatments and different approaches, such as invasive AC, stimulators of AC activity, PDE inhibitors, and cAMP analogs. The aim of this review is to comprehensively evaluate and provide data related to (i) the use of cAMP modulators during IVM and the effects on completion of meiosis and cytoplasmic reorganization, which are required for development of oocytes with the capacity to contribute to fertilization and subsequent embryonic development; and (ii) the main cAMP modulators and the effects when used in oocyte IVM.

Use of two doses of cloprostenol in different intervals for estrus synchronization in hair sheep under tropical conditions.

This study evaluated the effect of two doses of prostaglandin at different intervals on reproductive parameters of crossbred ewes. In Experiment 1, 30 ewes received two doses of 120 µg cloprostenol at 7 (G 7 days), 9 (G 9 days), or 11.5 (G 11.5 days) days apart. Ultrasound assessments were performed from the first and second cloprostenol administration for 5 days or ovulation detection. Estrus signs were checked by a teaser male. Plasma progesterone concentration was measured before each cloprostenol dose. In Experiment 2, 95 ewes were allocated into the same treatments and after the second dose, ewes in estrus were mated. At 30 days after breeding, pregnancy diagnosis was conducted and prolificacy was evaluated at lambing. In Experiment 1, at the first cloprostenol administration, 50% of ewes had an active CL and all showed estrus. At the second administration, 66.7% of ewes had an active CL and one did not present estrus. There was no difference (P > 0.05) after the second dose for as follows: overall estrous response (90%), interval from cloprostenol administration to estrous onset (42.0 ± 4.9 h), estrus duration (31.5 ± 2.1 h), ovulation rate (100.0%), and number of ovulations (1.5 ± 0.3). In Experiment 2, both pregnancy and prolificacy rates were similar (P > 0.05) for G 7 days (73.3; 145%), G 9 days (75.9; 125%), or G 11.5 days (75.9; 145%), leading to an overall pregnancy rate of 75.0% (66/88) and prolificacy rate of 137%. Therefore, the three treatments proposed were able to promote high pregnancy and prolificacy rates in crossbred ewes.

Effect of different hormonal combinations on follicular wave emergence and superovulatory response in sheep.

The aim of the present study was to compare hormonal treatments to induce and synchronize follicular wave emergence to improve the results of superovulatory (SOV) treatments in ewes. In Experiment 1 (n = 66), ewes were treated with a progesterone intravaginal implant plus a PGF2α analogue (group GP4), or with the same treatment plus estradiol benzoate (GP4+EB), a GnRH agonist (GP4+GnRH), or both, estradiol benzoate and a GnRH agonist (GP4+EB+GnRH) in a 2 × 2 factorial arrangement. Follicular wave emergence was determined by ultrasound. Follicular wave did not emerge during the studied period in 10 females (one from GP4, six from GP4+EB and three from GP4+EB+GnRH). Follicular emergence was less synchronized (P = 0.007) when estradiol was administered (GP4+EB: 103.6 ± 22.0 h), without any interaction with GnRH treatment (GP4+EB+GnRH: 80.1 ± 21.4 h, GP4+GnRH: 52.5 ± 8.7 h, GP4: 56.6 ± 10.4 h). Estradiol administration delayed the moment of follicular emergence (P = 0.007) and the follicular wave emergence moment in which follicular dominance was achieved (P = 0.009), without interactions between estradiol and GnRH in the moment of follicular wave emergence or dominance. In Experiment 2 (n = 22), two SOV protocols were compared: the best treatment of Experiment 1 (GP4) was used to synchronize follicular wave emergence, initiating the SOV treatment 2.5 days later; in the control treatment, SOV treatment started 80 h after a short-term protocol to synchronize ovulation (Gcontrol). The number of corpora lutea (CL) and the evaluation of the collected embryos were performed six days after estrus. Blood samples were collected daily for plasma progesterone determination. Although the number of CL was similar in Gcontrol (7.1 ± 1.0) and GP4 (6.9 ± 5.1), the number of structures and viable embryos recovered were greater in Gcontrol (P < 0.05). The occurrence of luteal premature regression was significantly greater in GP4 (60%) than in Gcontrol (8.3%). The use of GnRH agonist alone did not improve synchronization of follicular wave emergence. When EB was used (alone or associated) follicular wave emergence was less synchronized. The SOV protocol proposed had a similar ovarian response; however, it resulted in less transferable embryos.

Qualitative and quantitative analysis of bacteria from vaginitis associated with intravaginal implants in ewes following estrus synchronization / Análise qualitativa e quantitativa de bactérias da vaginite associadas com implante intravaginal em ovelhas após sincronização de estro

ABSTRACT: This study evaluated the presence of vaginitis and the bacterial load associated with different intravaginal implants in ewes. Twenty-four Dorper and crossbred ewes were allocated into three groups and received intravaginal implant containing 0.3g progesterone (CIDR(r)), 60mg MAP or sponges without progesterone (CONTROL) for six days. Then, CIDR and MAP treated-ewes received 12.5mg dinoprost and 300IU eCG. Vaginal mucus samples were collected at four times: before device insertion, at the day of its removal, 24 and 48 hours after. The samples were cultured and the colonies were counted (CFU/mL) and identified. The results obtained from the counting of CFU mL-1 were submitted to Kruskal-Wallis H test, with P<0.05 being considered significant. Before device insertion, 68.2% of the samples yielded Staphylococcus spp. and 60.0% of them were Staphylococcus aureus. After implant removal, 100% of ewes had clinical signs of vaginitis. However, the level of local infection in the CONTROL-ewes was lower (P>0.05) in comparison with MAP and CIDR-treated ewes. During the occurrence of vaginitis, the predominant isolates belonged to the coliform group, mainly Escherichia coli (72.7%). Such infection was not determined by the members of the vaginal microbiota that were present before implant insertion and normal microbiota was restored between 24 to 48 hours after insert removal.

RESUMO: Este estudo avaliou a presença de vaginite e contagem bacteriana associada ao uso de diferentes dipositivos intravaginais em ovelhas. Vinte e quatro fêmeas Dorper e mestiças foram alocadas em três grupos e receberam implante intravaginal contendo 0,3g de progesterona (CIDR(r)), 60mg de acetato de medroxiprogesterona (MAP) ou esponjas sem progesterona (CONTROLE) por seis dias. Posteriormente, as ovelhas tratadas com CIDR e MAP receberam 12,5mg de dinoprost e 300 UI de eCG. Amostras do muco vaginal foram coletadas em quatro momentos: antes da inserção do dispositivo, no dia de sua retirada, 24 e 48 horas após. As amostras foram cultivadas e as colônias foram contadas (UFC mL-1) e identificadas. Os resultados obtidos da contagem das UFC mL-1 foram submetidos ao teste do qui-quadrado, com P<0,05 sendo considerado significativo. Antes da inserção do dispositivo, 68,2% das amostras continham Staphylococcus spp., sendo 60,0% delas Staphylococcus aureus. Após a remoção do implante, 100% das ovelhas apresentaram sinais clínicos de vaginite. Entretanto, o grau de infecção local nas ovelhas do grupo CONTROLE foi menor (P>0.05), em comparação com as ovelhas recebendo CIDR e MAP. Durante a ocorrência da vaginite, os isolados predominantes pertenciam ao grupo dos coliformes, principalmente Escherichia coli (72,7%). Tal infecção não foi determinada pelos membros da microbiota vaginal, que estavam presentes antes da inserção dos dispositivos, e a microbiota normal foi reestabelecida entre 24 e 48 horas após sua remoção.

Efficiency of different hormonal treatments for estrus synchronization in tropical Santa Inês sheep.

The aim of this study was to evaluate the effect of (i) the duration of hormone treatment with progestogen sponges during the seasonal anestrus and (ii) the administration of two doses of prostaglandin at 7 days apart during the breeding season on reproductive parameters of Santa Inês ewes. In experiment 1, 32 ewes received intravaginal MAP sponges for 6 (G6 days), 9 (G9 days), or 12 (G12days) days and 75 µg D-cloprostenol i.m. and 300 IU eCG i.m. 1 day before sponge removal. In experiment 2, 23 ewes received two doses of 0.48-mg sodium cloprostenol i.m. 7 days apart. Ovarian follicular dynamic was assessed through transrectal ultrasonography. Blood samples were collected daily to determine progesterone concentrations. In experiment 1, estrus and ovulation rates did not differ (P > 0.05) among protocols and between cyclic and acyclic ewes at the beginning of the experiment. The G9 days treatment showed a lower dispersion of ovulations in relation to onset of estrus when compared to G6 days and G12 days. In experiment 2, all ewes exhibit estrus and ovulated after the second dose of prostaglandin, although ewes that were in diestrus at D0 showed subluteal concentrations of progesterone during the follicle development stage of the treatment. In conclusion, the use of progestogen device during 9 days promotes lower dispersion of ovulation when compared to its use for 6 or 12 days, and the protocol of two doses of prostaglandin 7 days apart synchronizes estrus efficiently but results in follicular development under low progesterone concentrations.