Review: Non-surgical artificial insemination and embryo recovery as safe tools for genetic preservation in small ruminants.

Artificial insemination (AI) and in vivo embryo production (or multiple ovulation and embryo transfer, MOET) programs are both instrumental in accelerating the propagation of genetically and economically superior goats and sheep. The aim of this review was to present the current gestalt of non-surgical AI and embryo recovery (NSER) procedures in small ruminants. Small body size, precluding rectal palpation, and highly limited penetrability of the uterine cervix in ewes are the major reasons for the scarce use of non-surgical assisted reproduction techniques in this species. As a result, AI and embryo recovery techniques in sheep mainly involve laparoscopy or laparotomy (LAP). In does, however, the Embrapa method of AI allows for successful intrauterine deposition of semen, resulting in pregnancy rates from 50 to 80% under field conditions (>3 000 goats inseminated) when frozen-thawed semen is used. After the administration of prostaglandin F2α (PGF2α), non-surgical (transcervical) embryo recovery is also feasible in goats, with the cervical penetration rate approaching 100%. There is a paucity of information on the efficacy of non-surgical AI using frozen semen in sheep, but the results are satisfactory with fresh, cooled, or chilled ram semen. An application of the NSER technique in ewes has greatly improved over the last decade, and cervical penetration rates of ∼90% can be achieved when a hormonal cervical dilation protocol using PGF2α, oxytocin, and/or estradiol ester (e.g., estradiol benzoate) is applied. In some genotypes of sheep, sufficient cervical dilation can be induced without estradiol ester included in the protocol. Several studies indicated that recovery of transferable quality ovine embryos using NSER is comparable to that employing a ventral midline laparotomy, and NSER is evidently a method of choice when animal welfare is concerned. Considering both the number of retrievable embryos and animal well-being, the NSER is a viable alternative for surgical procedures. With further developments, it has the makings of a primary, if not exclusive, embryo recovery technique in small ruminants worldwide.

Effects of different doses of estradiol benzoate used in a cervical relaxation protocol on the success of non-surgical embryo recovery and luteal function in superovulated ewes.

This study investigated the effectiveness of different doses of estradiol benzoate (EB) to promote cervical relaxation and their effects on luteal function and outcomes of non-surgical embryo recovery (NSER) in sheep. Multiparous (MULT) and nulliparous (NULL) crossbred Lacaune X Santa Inês ewes were superovulated and naturally bred. Seven days after progesterone withdrawal, females were randomly assigned to one of three distinct cervical relaxation protocols, consisting of i.m. treatment with 37.5 µg d-cloprostenol and different doses of EB: 0.0 mg (0.0EB group; n = 3 NULL and 14 MULT); 0.5 mg (0.5EB group; n = 4 NULL and 12 MULT) or 1.0 mg (1.0EB group, n = 6 NULL and 11 MULT) 16 h before NSER. All ewes received 50 IU of oxytocin 20 min before NSER (D17). Blood samples were collected and ultrasound exams (B-mode and color Doppler) were performed at two timepoints: immediately before d-cloprostenol and EB treatments and prior to NSER. Estrous behavior, corpora lutea count and NSER success outcomes were not affected by EB treatments nor parity (P > 0.05). Embryo recovery rate was greater for ewes in the 0.5EB group and in the NULL ewes (P < 0.05). Ovarian biometrics differed between the two evaluation timepoints in all groups (P < 0.05). Plasma estradiol increased over time, reaching a significant greater level in 1.0EB ewes compared to controls on D17 (P < 0.05), whereas progesterone concentrations decreased over time in all groups (P > 0.05). In conclusion, treatments did not affect NSER success but they did affect luteal function by altering P4 and E2 concentrations. Therefore, the NSER technique can be successfully performed in ewes with or without prior treatment with EB.

Single dose of 300 IU hCG in the early luteal phase in superovulated ewes: Effects on corpora lutea, progesterone profile, and embryo recovery.

This study aimed to evaluate the effects of hCG treatment during the early luteal phase on ovarian function, progesterone profile, and embryo yield in superovulated ewes. Superovulated sheep were randomly assigned to receive 300 IU hCG i.m. (GhCG, n = 24) or not (GControl, n = 25) at 96 h after the removal of the progesterone (P4) device (D13). Non-surgical embryo recovery (NSER) was performed eight days after P4 withdrawal. Ultrasound evaluations were performed on D13, D14, D16, and D17. Blood samples were collected on D14, D16, and D17. Superovulation scores were recorded based on the number of corpora lutea (CL) as follows: 1 (≤ 2), 2 (3-5), 3 (6-8), and 4 (≥ 9). NSER efficiency, superovulation response, and luteal tissue area were similar in both groups (P > 0.05). Structural luteolysis tended to be higher in GControl (P = 0.07; 47.0 %) while functional luteolysis was similar in both groups (P > 0.05; 0.0 % and 5.9 %). The recovery rate was greater (P < 0.05) in GhCG (89.8 %) compared with GControl (71.0 %), with similar overall ova/embryo numbers observed for both groups (P > 0.05). GhCG showed a higher concentration of animals with a superovulatory response score of 4 (54.5 %; P < 0.05) compared with the lowest scores. Plasma progesterone on D16 was higher (P < 0.05) in GhCG ewes (11.1 ± 1.5 vs 6.9 ± 1.5 ng/mL). In conclusion, the hCG treatment improved circulating P4 and embryo recovery rate, tended to maintain luteal functionality, and thus constitutes an additional tool for improving embryo yield in superovulated ewes.

Exogenous progestogens differentially alter gene expression of immature cumulus-oocyte complexes in sheep.

This study evaluated the role of progesterone (P4) and medroxyprogesterone acetate (MAP) on the molecular status of immature cumulus-oocyte complexes (COCs) and the implications for oocyte quality in sheep. The number of viable COCs per ewe and the rate of COCs screened for developmental competence by brilliant cresyl blue positive (BCB+) were similar (P > 0.05), respectively, across treatments (P4: 7.7 ± 0.7 and 4.7 ± 1.2; MAP: 5.7 ± 1.0 and 3.5 ± 2.3; and control: 5.7 ± 1.1 and 3.6 ± 2.4). The COCs' gene expression was altered by exogenous progestogens compared with the control group: markers of steroidogenic pathway (FSH receptor [FSHr], LH receptor [LHr], and estradiol receptor α) and of quality (zygote arrest 1, growth differentiation factor 9, and B-cell lymphoma 2) were in abundance in P4 (P < 0.05). In addition, reelin protein (RELN) was downregulated, and Bcl-2 was upregulated in MAP (P < 0.05). In the P4 vs MAP comparison, FSHr, LHr, and RELN genes were upregulated (P < 0.05) in the P4 group. In conclusion, P4 and MAP promoted dissimilar effects on transcriptome profiling of immature BCB-selected COCs, possibly due to the differences in the chemical structure of progestogens and concentrations of serum P4. Exogenous P4 impacted positively on the profile of genes related to oocyte quality.

Administration of a single dose of 300 IU of human chorionic gonadotropin seven days after the onset of estrus improves pregnancy rate in dairy goats by an unknown mechanism.

This study examined the effects of exogenous hCG administration on ovarian function and pregnancy rates in estrous-induced dairy goats during the transition into the breeding season. Eighty-six Toggenburg does received 60 mg of medroxyprogesterone acetate intravaginal sponge for 6 d plus 200 IU of equine chorionic gonadotropin and 30 µg of d-cloprostenol i.m. 24 h before sponge removal, and were then bred for 96 h. Seven days (D7) after first mating the does received either 1 mL of saline (the control group, n = 43) or 300 IU of hCG (the hCG-treated group, n = 43) i.m. Transrectal ovarian ultrasonography (B-mode and color Doppler) was performed on D7, D13, D17, and D21 and ultrasonographic pregnancy detection on D30. Pregnancy rate was higher (P < 0.05) in hCG-treated goats (90.7%; 39/43) than that in control animals (74.4%; 32/43). Accessory luteal structures (ALSs) were detected in 46.5% (20/43) of hCG-treated does. All hCG-treated does that had ALSs and 82.6% of goats without ALS post-treatment remained pregnant. The total luteal area increased (P < 0.05) from D7 to D13 in pregnant animals of both groups, whereas mean vascular area declined (P < 0.05) by D21 in all nonpregnant does. Serum progesterone concentrations increased (P < 0.05) on D21 in pregnant goats of both groups, but they were related to changes in luteal tissue content only in control does throughout the present study. Mean daily numbers of small- and medium-sized antral follicles decreased (P < 0.05) only in pregnant animals of both groups with a decline in medium follicle numbers occurring earlier in hCG-treated (D13) compared with control does (D17). To summarize, a single dose of hCG given on D7 after estrus was followed by a decrease in the number of medium-sized antral follicles in gestating hCG-treated does, induced the formation of ALSs in ~47% of all hCG-treated does, and significantly increased the pregnancy rate in estrous-induced Toggenburg goats in the transition to the breeding season.

Exogenous progestogen does not affect first-wave follicle populations and oocyte quality during ovarian stimulation with FSH in sheep.

The effect of short-term administration of medroxyprogesterone acetate (MPA) or natural progesterone (P4) during ovarian stimulation with FSH on oocyte recovery was investigated in Santa Inês ewes. Ewes were treated with an intravaginal sponge containing MPA for 6 d; GnRH was applied 36 h after sponge removal and FSH was given in 3 injections (40, 24, and 16 mg, respectively) every 12 h after (D0, approximate ovulation time). At the first FSH dose, the ewes received either a new MPA sponge (n = 10) or a controlled device for internal release impregnated with P4 (n = 10) or did not receive any device (n = 10). Ovarian dynamics were assessed every 12 h by transrectal ultrasonography from D-3 to D2. Oocytes were recovered by laparoscopic ovum pick-up (LOPU) on D2 and graded by morphologic quality. The number of small, medium, and large follicles at D0 and D2 (ultrasound examinations), number of both follicles aspirated and oocytes recovered at LOPU, recovery rate, and oocyte grade did not differ (P > 0.05) among treatments. Thus, the short-term use of MPA or P4 during ovarian stimulation did not affect the first-wave follicle population or morphologic quality of oocytes. We would suggest that, in this protocol, the use of exogenous progestin is unnecessary.

Mini-percoll gradient may be used for frozen-thawed sperm selection in sheep / Gradiente de mini-Percoll pode ser utilizado na seleção espermática de sêmen congelado de ovinos

This study evaluated the effect of increasing centrifugal force and reducing centrifugation time and volume in Percoll protocols on ram sperm parameters. Commercial semen of Santa Inês rams were used and five treatments were performed: traditional Percoll and mini-Percoll (MP) techniques (I- 5000 x g, 5min; II- 2500 x g, 5min; III- 1250 x g, 5min; IV- 700 x g, 10min). At post-thawing (PT) and post-selection protocols (0h), samples were assessed for spermatozoa recovery rate, motility, plasma membrane (PM) integrity, sperm capacitation and morphology and incubated at 37 C for 1, 2 and 3h. The sperm recovery rate averaged 9.1±1.4%, and most motility parameters were similar (P> 0.05) among protocols. VCL (µm/s) was higher (P< 0.05) after MP-II, III and IV (66.1±4.5) than traditional Percoll (46.3±4.9). Capacitation status and PM integrity were similar (P> 0.05) among treatments. For the first time, we have demonstrated the reduction of the gradient volume and centrifugation time associated with an increase on centrifugation force at Percoll can be successfully used for frozen-thawed ram sperm selection. MP may be used instead of traditional Percoll, decreasing costs and semen handling time.(AU)

O presente estudo avaliou o efeito do aumento da força de centrifugação, bem como da redução do tempo de centrifugação e do volume do gradiente de Percoll em diferentes protocolos nos parâmetros espermáticos de ovinos. Foi utilizado sêmen comercial de carneiros da raça Santa Inês, e cinco tratamentos foram realizados: Percoll tradicional e quatro técnicas de mini-Percoll (I- 5000 x g, 5min; II- 2500 x g, 5min; III- 1250 x g, 5min; IV- 700 x g, 10min). Após o descongelamento e a seleção espermática em cada técnica utilizada (0h), amostras foram avaliadas quanto à taxa de recuperação espermática, motilidade, integridade de membrana plasmática, capacitação e morfologia. Ao final, foram incubadas a 37 ºC por uma, duas e três horas. A taxa de recuperação média (9,1±1,4%) e a maioria dos parâmetros de motilidade foram similares (P>0,05) entre os tratamentos. VCL foi maior (P<0,05) após MP-II, III e IV (66,1±4,5) quando comparados ao Percoll tradicional (46,3±4,9). O status da capacitação e a integridade de membrana foram similares (P>0,05) entre os tratamentos. Pela primeira vez, foi demonstrado que a redução do volume do gradiente utilizado e do tempo de centrifugação, associada com o aumento da força de centrifugação nos protocolos de Percoll, pode ser usada com sucesso na seleção espermática de sêmen congelado de ovinos. O mini-Percoll pode ser utilizado em alternativa à técnica de Percoll tradicional, diminuindo custos e tempo de manipulação do sêmen durante a técnica.(AU)

Cervical penetration rates and efficiency of non-surgical embryo recovery in estrous-synchronized Santa Inês ewes after administration of estradiol ester (benzoate or cypionate) in combination with d-cloprostenol and oxytocin.

The effects of estradiol esters, d-cloprostenol and oxytocin on induction of cervical dilation prior to non-surgical embryo recovery in Santa Inês ewes (Days 6-7 estrous cycle) were assessed in this study. In Trial 1, transcervical embryo flushing was performed in estrous-induced ewes administered 37.5 µg of d-cloprostenol i.m. 10 h before and 50 IU of oxytocin i.v. 20 min before uterine flushing with (EB-PGF-OT; n = 13) or without (PGF-OT; n = 11) 1 mg of estradiol benzoate i.m. administered concurrently with d-cloprostenol injection. In Trial 2, the estrous-synchronized animals were treated with 1 mg of estradiol benzoate (EB-PGF-OT; n = 12) or estradiol cypionate (EC-PGF-OT; n = 12) i.m. along with 37.5 µg of d-cloprostenol i.m. 16 h before and 50 IU of oxytocin i.v. 20 min before uterine flushing. In Trial 1, uterine flushing could be accomplished in 38% of ewes in the EB-PGF-OT and 27% those in the PGF-OT (P>0.05) group. Flushing fluid recovery averaged 90% and there were 1.0 ± 1.1 embryos/ewe collected with mean duration of the flushing procedure being ˜36 min. In Trial 2, uterine flushing was accomplished in 78% of ewes in the EB-PGF-OT and 44% of those in the EC-PGF-OT group (P>0.05) with mean flushing fluid recovery rate being 88% and time elapsing to complete flushing being ˜33 min. Within the subsets of animals treated with EB, the percentages of successful transcervical penetrations were 38% compared with 78% in Trials 1 and 2, respectively (i.e., with EB administered 10 h compared with 16 h before uterine flushing: P<0.05). The interval from EB administration to the beginning of transcervical penetration can affect the efficacy of embryo recovery procedures utilizing a combined EB/d-cloprostenol/oxytocin pre-treatment.

Use of human intravaginal tampon embedded with natural progesterone induces synchronous estrus in Santa Inês ewes / Uso de tampão intravaginal humano embebido com progesterona humana para indução e sincronização de estro em ovelhas Santa Inês

Este estudo avaliou a eficiência de três protocolos de indução de estro síncronizado em ovelhas da raça Santa Inês. Vinte e quatro ovelhas adultas foram equitativamente distribuídas em três grupos, de acordo com ordem de parto, peso corporal (kg) e escore da condição corporal. As ovelhas receberam implante vaginal de progesterona natural por seis dias mais 37,5µg de d-cloprostenol laterovulvar e 300UI de eCG i.m., 24 horas antes da remoção do dispositivo. Ovelhas controle receberam CIDR330mg de progesterona, e as demais receberam dispositivo absorvente intravaginal humano, tamanho mini, embebido com 200 (OB200) ou 400mg (OB400) de progesterona. Coletas de sangue foram feitas nos momentos D0 (antes da inserção dos dispositivos), D0+6h e diariamente, até um dia após retirada do dispositivo (D7). A progesterona (ng/mL) foi semelhante (P>0,05) em todos os tratamentos ao longo do período experimental, exceto no dia da remoção do dispositivo, quando as ovelhas controle (2,5±0,3) tiveram progesterona superior (P<0,05) às ovelhas OB200 (0,6±0,1) e OB400 (1,2±0,4). O percentual de animais em estro (100,0, 62,5 e 100,0%) foi semelhante (P>0,05) e o intervalo para o estro (46,3±3,9a, 26,4±4,5b e 31,2±5,8a,b) foi diferente (P<0,05) entre os animais dos grupos controle, OB200 e OB400, respectivamente. A taxa de concepção das ovelhas foi de 50,0% (4/8) para cada grupo (P>0,05). Dispositivos humanos e fonte de progesterona podem ser usados para induzir o estro sincronizado em ovelhas Santa Inês.(AU)

Hydrometra in dairy goats: Ultrasonic variables and therapeutic protocols evaluated during the reproductive season.

Hydrometra is characterized by the accumulation of fluid within the uterus due to the persistence of corpus luteum. The diagnosis of this disorder occurs with an ultrasonic exam. This study evaluated uterine drainage and fertility rates in goats after the use of d-cloprostenol in association or not with Gonadotropin-releasing hormone (GnRH) treatment. Twenty Saanen goats, diagnosed with hydrometra, received three 37.5-µg doses of d-cloprostenol laterovulvarly at 10-day intervals. On D5, the goats were assigned into two groups receiving 1 mL of GnRH or saline solution intramuscularly. Ultrasonography (US) was performed from D0 to D25. An US approach was used to rank hydrometra in scores. The pregnancy rate was assessed 45 and 90 days after the end of treatment. The uterine fluid was totally drained after the first and second administration of d-cloprostenol in 50% and 95% of the goats, respectively. In one female, full emptying of the uterus occurred only after D20. US performed at 45 and 90 days after the end of treatment indicated there was a pregnancy rate of 45.0% and 55.0%, respectively. Fertility did not differ between the GnRH-treated and control goats. Those goats not pregnant at 45 days had a follicular cyst, hydrosalpinx or hydrometra. At 90 days, no change was observed in the hydrosalpinx, and four goats had hydrometra. The use of three doses of d-cloprostenol 10 days apart was efficient for induction of draining the contents of the uterus, resulting in a relatively acceptable pregnancy rate. This treatment associated with the US approach can be important when applied in the field.

Transrectal ultrasound evaluation in tropical dairy goats: an indispensable tool for the diagnosis of reproductive disorders.

In Brazil, great milk productivity was achieved after the implementation of a genetic improvement program. However, reproductive efficiency is still far from optimal, possibly due to the high number of undiagnosed disorders that may affect fertility. The aim of this study was to evaluate occurrences of the main reproductive disorders in dairy goats in southeastern Brazil. Data were collected between January 2015 and May 2017 from 23 commercial herds of different breeds, with goats ranging from 8 months to 12 years of age. Transrectal ultrasound exams were performed in 2680 goats. A total of 14.8% of the does showed a disorder in the reproductive tract: hydrometra (10.0%), ovarian follicular cysts (2.3%), gestational loss (1.5%), and hydrosalpinx (1.1%). This was the first study evaluating reproductive disorders in live animals that used a high number of Brazilian dairy goats. Considering that all these diseases affect fertility to different degrees, the performance of transrectal ultrasonography exams twice a year is strongly suggested, in order to guide precocious treatment or discard the animal as soon as possible, thus reducing economic losses in dairy goat farming.

Cervical relaxation for non-surgical uterus access in Santa Inês ewes / Relaxamento cervical para acesso uterino não cirúrgico em ovelhas Santa Inês

The present study was composed by two experiments aiming to develop a cervical dilation technique for non-surgical access to Santa Inês ewe's uterus. In Experiment 1, thirty ewes underwent four epidural treatments. The three experimental treatments used 2.0mg/kg ketamine. The group receiving this drug alone was denominated KG, whereas other group had ketamine associated with 0.1mg/kg morphine (KM) and KX a third group had ketamine associated with 0.05mg/kg xylazine (KX). Control treatment was 1mL/7.5kg saline solution epidurally (CON). Cervical dilation was evaluated in both experiments by attempting to pass a metal rod through the cervix. In Experiment 2, three different hormonal protocols for cervical dilation were tested in thirty ewes. Epidural anesthesia with 2.0mg/kg ketamine was the control treatment (KG) and was combined with hormonal treatments: Misoprostol (MI); Oxytocin + Estradiol (OE); Misoprostol + Oxytocin + Estradiol (MOE). In Experiment 1 transposition rate was not different among groups. In Experiment 2, OE presented the highest rate (90%) while MOE presented 86.2%, MI 68.9% and CON 62.1%. The study developed a pharmacological protocol that increased cervical transposition making the non-surgical access to the uterus feasible in Santa Inês ewes.(AU)

O presente estudo teve como objetivo desenvolver uma técnica para acesso não cirúrgico ao útero de ovelhas Santa Inês e foi realizado em duas etapas. No experimento 1, 30 ovelhas foram submetidas a quatro tratamentos epidurais. Os três tratamentos teste usaram 2,0mg/kg de cetamina. O grupo que recebeu apenas cetamina foi denominado KG, enquanto no segundo grupo a cetamina foi combinada com morfina (0,1mg/kg - KM) e um terceiro grupo recebeu cetamina associada com xilazina 0,05mg/kg - KX). No grupo controle (CON), usou-se solução salina (1mL/7,5kg de peso). A dilatação cervical foi avaliada em ambos os experimentos pela tentativa de transposição cervical com uma haste metálica. No experimento 2, três protocolos hormonais de dilatação cervical foram testados em 30 ovelhas. Anestesia epidural com 2,0mg/kg de cetamina foi o tratamento controle (CON), combinada com tratamentos hormonais nos grupos: misoprostol (MI); ocitocina + estradiol (OE); misoprostol + ocitocina + estradiol (MOE). No experimento 1, a taxa de transposição não variou entre os grupos. No experimento 2, OE teve a maior taxa (90%), enquanto MOE apresentou 86,2%, MIi 68,9% e CON 62,1%. O estudo desenvolveu um protocolo farmacológico que aumentou a taxa de transposição cervical, tornando o acesso uterino não cirúrgico viável em ovelhas Santa Inês.(AU)

Freezing goat embryos at different developmental stages and quality using ethylene glycol and a slow cooling rate / Congelação de embriões caprinos em diferentes estádios de desenvolvimento e qualidade utilizando etileno glicol e taxa de resfriamento lenta

The efficiency of an alternative freezing protocol for goat embryos of different morphology and quality was tested. Fifty-eight embryos on Day 6-7 stage were transferred as fresh or after freeze-thawing (n=29/group). For freezing, embryos were placed into 1.5M ethylene-glycol solution for 10min. During this time, they were loaded in the central part of 0.25mL straw, separated by air bubble from columns containing PBS/BSA 0.4% plus 20% BFS. Straws were then frozen using a freezing machine from 20ºC to -6ºC at a cooling rate of 3ºC/min, stabilization for 15min (seeding after 5min), from -6 C to -32ºC at 0.6 C/min,and plunged into liquid nitrogen. Frozen embryos were thawed for 30s at 37ºC in a water bath. Embryos subjected to fresh transfer were maintained in holding medium (37ºC). Fresh and frozen-thawed embryos were transferred at day 7 post-estrus to 30 recipients. Kidding and kid born rates were similar (P> 0.05), respectively, for recipients receiving fresh (66.7% or 10/15; 55.2% or 16/29) or frozen-thawed (60% or 9/15; 51.7% or 15/29) embryos. The cryopreservation of goat embryos using slow-freezing protocol and 1.5MEG resulted in similar efficiency rates of fresh embryos.(AU)

Este estudo testou a eficiência de protocolo alternativo de criopreservação de embriões caprinos de diferentes qualidades morfológicas. Foram utilizados 58 embriões, coletados entre o sexto e o sétimo dia do ciclo estral (n=29/grupo). Embriões congelados passaram por solução 1,5M etilenoglicol por 10min e foram aspirados durante esse tempo para parte central de palheta 0,25mL, separada por bolhas de ar de colunas contendo PBS 0,4% BSA e 20% SFB. As palhetas foram congeladas em máquina de congelação de 20ºC a -6ºC, com taxa de resfriamento de 3ºC/min, estabilização por 15min (seeding após 5min), -6ºC a -32ºC a 0,6ºC/min, e imersas em nitrogênio líquido. Os embriões foram descongelados por 30s a 37ºC, em água. Embriões frescos foram mantidos em solução de manutenção (37ºC). Embriões frescos e congelados/descongelados foram transferidos para 30 receptoras no sétimo dia do ciclo estral. A taxa de partos e a de crias nascidas (respectivamente) foram similares (P>0,05) para receptoras recebendo embriões frescos (66,7% ou 10/15; 55,2% ou 16/29) ou congelados/descongelados (60,0% ou 9/15; 51,7% ou 15/29). O protocolo de criopreservação de embriões utilizado no presente estudo resultou em índices de eficiência semelhantes aos de embriões frescos.(AU)

Potential role for GnRH in the synchronization of follicular emergence before the superovulatory Day 0 protocol.

The ability of gonadotropin-releasing hormone (GnRH) to synchronize ovulation and new follicular wave emergence before a "superovulatory Day 0" protocol was assessed in Santa Inês ewes. For estrus synchronization, a 60-mg medroxyprogesterone acetate sponge was inserted for 6 d. One day before sponge removal, 37.5-µg d-cloprostenol and 300 IU equine chorionic gonadotropin were injected intramuscularly (i.m.). After sponge removal, ewes were assigned to the following 3 groups: (1) GC-1 mL saline at 12 h (n = 10); (2) G24h-0.025-mg lecirelin (GnRH agonist) i.m. at 24 h (n = 10); or (3) G36h-0.025-mg lecirelin i.m. at 36 h (n = 9). Ovarian ultrasonography was conducted to assess follicular dynamics. Blood was collected to determine plasma concentrations of progesterone and estradiol. Females from G36h and GC had a greater (P < 0.05) estrous response than those from the G24h group (78.0 and 90.0 vs 0.0%, respectively). Ewes from G24h and G36h had earlier (P < 0.05) ovulation (48.0 ± 10.2 and 56.7 ± 5.7 h) compared with those from Gc (64.1 ± 9.7 h). The mean number of ovulations per ewe was greater (P < 0.05) in Gc (1.9 ± 0.6) and G36h (2.0 ± 1.0) than G24h (1.2 ± 0.4). Plasma concentrations of progesterone and estradiol differed over time. Follicular growth during the postovulatory day was affected (P < 0.05) by day of the estrus cycle as well as by the interaction (P < 0.05) of treatment and day of the estrus cycle. There was a larger (P < 0.05) population of medium follicles during the first 24 h after the ovulation in G24h compared with Gc, and there was an absence of large follicles in G36h between 36 and 72 h after ovulation. In conclusion, the use of GnRH agonist at 36 h more efficiently synchronized ovulation and promoted the absence of dominant follicles during early diestrus and may be used at the start of superovulatory treatment at 80 h in Santa Inês ewes.

Comparative analysis of laparoscopic and ultrasound-guided biopsy methods for gene expression analysis in transgenic goats.

The present study aimed to compare laparoscopic (LP) and ultrasound-guided (US) biopsy methods to obtain either liver or splenic tissue samples for ectopic gene expression analysis in transgenic goats. Tissue samples were collected from human granulocyte colony stimulating factor (hG-CSF)-transgenic bucks and submitted to real-time PCR for the endogenous genes (Sp1, Baff, and Gapdh) and the transgene (hG-CSF). Both LP and US biopsy methods were successful in obtaining liver and splenic samples that could be analyzed by PCR (i.e., sufficient sample sizes and RNA yield were obtained). Although the number of attempts made to obtain the tissue samples was similar (P > 0.05), LP procedures took considerably longer than the US method (P = 0.03). Finally, transgene transcripts were not detected in spleen or liver samples. Thus, for the phenotypic characterization of a transgenic goat line, investigation of ectopic gene expression can be made successfully by LP or US biopsy, avoiding the traditional approach of euthanasia.