Abstract Cryoprotectants are required to reduce damage caused to the cells due to low temperatures during the cryopreservation. Antifreeze proteins (AFP) have a well-known role in cell membrane protection, while resveratrol is a potent antioxidant. This study assessed the effect of the association of resveratrol concentrations and AFP I in a ram semen extender. Pooled semen of four rams was allocated into six treatments in a factorial arrangement: (CONT, only the semen extender); only AFP I (ANT: 0.1 µg/mL of AFP I), only resveratrol, one treatment with two levels (10 µM/mL or 50 µM/mL of resveratrol); and two treatments with the interactions, with one AFP I and one of the two levels of resveratrol (0.1 µg/mL of AFP I with 10 µM/mL resveratrol; 0.1 µg/mL of AFP I with 50 µM/mL resveratrol). No interaction between factors was observed on sperm kinetics, plasma membrane integrity, hypo-osmotic test, and mitochondrial activity parameters. There was a high probability (P = 0.06) of reducing sperm cells with functional membrane percentage in the hypo-osmotic test and increasing the percentage of sperm with high mitochondrial activity (P = 0.07) was observed in AFP presence. An interaction of AFP and resveratrol was observed in non-capacitated sperm (P = 0.009), acrosomal reaction (P = 0.034), and sperm binding (P = 0.04). In conclusion, the association of resveratrol and AFP did not improve the quality of frozen-thawed semen and even promoted deleterious effects compared to their single addition in the semen extender. The supplementation of 50 µM/mL of resveratrol improved the outcomes of frozen-thawed ram sperm, being a potential cryoprotectant. cryopreservation (AFP wellknown well known protection antioxidant extender arrangement CONT, CONT (CONT extender) ANT (ANT 01 0 1 0. µgmL µg mL I, , I) (1 µMmL µM 5 resveratrol) interactions (0. resveratrol. . kinetics integrity hypoosmotic hypo osmotic parameters P 0.06 006 06 0.07 007 07 presence noncapacitated non capacitated 0.009, 0009 0.009 009 0.009) 0.034, 0034 0.034 034 0.034) 0.04. 004 0.04 04 0.04) conclusion frozenthawed frozen thawed cryoprotectant ( (0 0.0 00 000 0.00 003 0.03 03

Abstract Oviduct fluid extracellular vesicles (oEV) are essential for periconceptional events. The presence of EV has already been identified in the oviduct fluid (OF) from mammalian species, except in caprine. Therefore, this study aimed to isolate and characterize the caprine oEV (coEV). Initially, in Experiment 1, coEV were isolated from the OF of either each animal individually or from a pool of three animals. In experiment 2, coEV were isolated during the follicular or luteal phases of the estrous cycle. The coEV were characterized by size distribution, polydispersity index (PDI), and zeta potential using dynamic light scattering (DLS) analysis, as well as, by transmission electron microscopy (TEM) and dot blotting (DB). Our results indicated that the physicochemical characteristics of the coEV were similar (P > 0.05), regardless of the isolation method (individual or pool). However, coEV collected during the luteal phase were larger (P < 0.05) than those during the follicular phase. The TEM showed spherical and cup-shaped particles, characteristic of exosomes. The DB revealed the presence of exosomal proteins involved in the biogenesis of coEV. In conclusion, it is possible to isolate and characterize coEV from a single caprine female and the estrous cycle phase influences the vesicles average size and PDI. (oEV events (OF species Therefore . (coEV) Initially 1 animals 2 distribution PDI, PDI , (PDI) DLS (DLS analysis (TEM DB. (DB) P 0.05, 005 0.05 0 05 individual pool. pool) However cupshaped cup shaped particles exosomes conclusion (coEV (PDI (DB 00 0.0 0.

Abstract In vitro embryo production (IVEP) is an extremely important tool for genetic improvement in livestock and it is the biotechnology that has grown the most recently. However, multiple ovulation followed by embryo transfer is still considered the leading biotechnology for embryo production in small ruminants. This review aimed to identify what is still missing for more efficient diffusion of IVEP in small ruminants, going through the IVEP steps and highlighting the main factors affecting the outcomes. Oocyte quality is essential for the success of IVEP and an aspect to be considered in small ruminants is their reproductive seasonality and strategies to mitigate the effect of season. The logistics for oocyte collection from live females is more complex than in cattle, and tools to simplify this collection system and/or to promote an alternative way of recovering oocytes may be an important point in this scenario. The heterogeneity of oocytes collected from growing follicles in live females or from ovaries collected from abattoirs remains a challenge, and there is a demand to standardize/homogenize the hormonal stimulatory protocols and IVM protocols for each source of oocytes. The use of sexed semen is technically possible, however the low market demand associated with the high costs of the sexing process prevents the routine use of this technique, but its higher availability is an important aspect aiming for greater dissemination of IVEP. New noninvasive approaches for embryo selection are key factors since the selection for transfer or cryopreservation is another difficulty faced among laboratories. Embryo selection is based on morphological traits, although these are not necessarily reliable in predicting pregnancy. Several issues described in this review must be considered by researchers in other to promote the diffusion of IVEP in small ruminants. (IVEP recently However outcomes season cattle andor scenario challenge standardizehomogenize standardize homogenize possible technique laboratories traits pregnancy

RESUMO: Ainda não há consenso sobre qual é o protocolo mais apropriado para a produção in vivo de embriões em ovinos, apesar do crescente conhecimento. Uma abordagem para mitigar a variabilidade de resposta de ovelhas à superovulação (SOV) é iniciar a aplicação de gonadotrofinas em uma condição ovariana favorável. O presente estudo comparou três tratamentos em delineamento do tipo crossover: protocolo de SOV tradicional (TRAD) e “Dia 0” D0 SOV sem (D0-GnRH) ou com GnRH (D0+GnRH). Quinze ovelhas Santa Inês foram superovuladas com 200 mg de FSH em seis doses decrescentes e receberam PGF2α na quinta dose de FSH. As ovelhas foram submetidas a monta natural com carneiros férteis e os embriões colhidos por via cirúrgica. O número de embriões viáveis não diferiu entre os tratamentos (TRAD = 6,0 ± 4,7; D0-GnRH = 3,8 ± 6,4; D0+GnRH = 7,5 ± 6,5). Independentemente do tratamento, ovelhas com folículos ≤ 4 mm na primeira dose de FSH produziram mais embriões viáveis (9,6 ± 6,0; P < 0.05) quando comparadas aos animais que apresentavam folículos > 4 mm no início da SOV (2,9 ± 3,1 embriões viáveis). Os grupos TRAD e D0+GnRH apresentaram menor número de animais com folículos grandes (> 4 mm), no momento da primeira dose de FSH, quando comparados ao grupo D0-GnRH (P < 0,05). Em conclusão, os protocolos TRAD e D0+GnRH induziram uma condição ovariana mais favorável (folículos ≤ 4 mm) para a SOV. No entanto, os três tratamentos apresentaram eficiência semelhante em ovelhas Santa Inês.

ABSTRACT: There is still no consensus regarding the best protocol for in vivo embryo production in sheep despite increasing studies in this area. Moreover, there is variability in the response of ewes to superovulation (SOV). An approach to mitigate this inconsistency is to initiate gonadotropin administration under favorable ovarian conditions. The present study compared three treatments in a crossover design: a traditional SOV protocol (TRAD) and “Day 0” D0 SOV protocol with (D0+GnRH), or without Lecilerin (D0-GnRH). Fifteen Santa Inês ewes received 200 mg of FSH at six decreasing doses and PGF2α with the fifth dose of FSH. They were naturally mated with fertile rams and subjected to surgical embryo collection. The number of viable embryos was similar among the different treatments (TRAD = 6.0 ± 4.7; D0-GnRH = 3.8 ± 6.4; D0+GnRH = 7.5 ± 6.5). Regardless of the treatment method, ewes with follicles ≤ 4 mm, at the first FSH dose, produced more viable embryos (9.6 ± 6.0, P < 0.05) compared to ewes that had follicles > 4 mm at the beginning of the SOV (2.9 ± 3.1, viable embryos). Both the TRAD and D0+GnRH groups had fewer animals with large follicles (> 4 mm) at the first FSH dose than the D0-GnRH group (P < 0.05). In conclusion, both the TRAD and D0+GnRH treatments induced a more favorable ovarian condition (follicles ≤ 4 mm) for adequate SOV; although, all three treatments exhibited similar efficacies in Santa Inês sheep.

Resumo Este estudo avaliou os efeitos da dominância social sobre o comportamento do estro e os parâmetros ovarianos de cabras leiteiras submetidas à indução sincronizada do estro durante a estação não reprodutiva. A sincronização foi induzida em 23 cabras leiteira alocadas em baias coletivas. A ultrassonografia foi realizada em intervalos de 12 horas desde a remoção do dispositivo até a ovulação. Após a detecção do início do estro (EO), as cabras foram imediatamente removidas dos currais. A dominância foi pontuada de acordo com EO em relação à remoção do dispositivo, sendo: HD - alto dominante (<36 h; n = 8), MD - médio dominante (36 a 48 h; n = 10) e LD - baixo dominante (>478 h; n = 5). As cabras em estro foram inseminadas artificialmente em tempo flexível (FxTAI). O intervalo de estro foi maior (P<0,05) em LD (53,7 ± 6,5 h) do que em MD (37,9 ± 5,5 h) e cabras HD (32,3 ± 6,6 h), por sua vez, o intervalo entre a remoção do dispositivo e a ovulação foi semelhante (P> 0,05) em todos os grupos. O intervalo de EO a FxTAI foi maior em cabras HD quando comparado aos outros grupos (P<0,05). Em conclusão, embora a hierarquia social tenha influenciado o tempo de início do estro, os parâmetros ovarianos incluindo a ovulação não foram afetados.

Abstract This study assessed the effects of social dominance on the behavioral estrus and ovarian parameters of dairy goats subjected to synchronous estrus induction during the non-breeding season. Synchronous estrus was induced in 23 dairy goats allocated to collective pens. Ovarian ultrasonography was performed at 12 h intervals from device removal to ovulation. Upon estrus onset (EO) detection, the goats were immediately removed from the pens. Dominance was scored according to EO in relation to device removal in each pen, with HD - high dominants (<36 h; n = 8), MD - medium dominants (36 to 48 h; n = 10) and LD - low dominants (>48 h; n = 5). Goats in estrus underwent flexible time artificial insemination (FxTAI) according to EO. The estrus response was 100.0%. The interval to estrus was longer (P<0.05) in LD (53.7 ± 6.5 h) than in MD (37.9 ± 5.5 h) and HD goats (32.3 ± 6.6 h), while the interval from device removal to ovulation was similar (P>0.05) among all groups. The interval from EO to FxTAI was longer in HD goats compared to the other groups (P<0.05). In conclusion, although social hierarchy influenced the time of EO, the ovarian parameters, including ovulation, were not affected.

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.

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.