Effect of antifreeze protein I in the freezing solution on in vivo-derived sheep embryos.

This study evaluated the effects of different antifreeze protein type I (AFP I) concentrations added to a slow freezing solution in sheep in vivo-derived embryos. Good-quality embryos were allocated into: AFP-free (CONT); 0.1 µg/mL of AFP I (AFP0.1); or 0.5 µg/mL of AFP I (AFP0.5). After thawing, embryos were in vitro cultured (IVC) for 48 h. At 24 h and 48 h of IVC, dead cells and apoptosis, mitochondrial activity, intracellular reactive oxygen species (ROS), and glutathione (GSH) evaluations were performed. At 24 h, evaluated embryos were submitted to RT-qPCR for metabolism (SIRT2, PRDX1, OCT4, CDX2) and quality (AQP3, CDH1, HSP70, BAX, BCL2) genes. The in vitro survival rate was 56% (22/39) for CONT, 60% (32/53) for AFP0.1, and 53% (23/43) for AFP0.5 (p > 0.05). A tendency (p = 0.09) for a higher blastocyst hatching rate was noted in AFP0.1 (62%) compared to AFP0.5 (33%), and both groups were similar to CONT (50%). An increased (p < 0.05) mitochondrial activity at 24 h was observed in AFP0.1 compared to CONT. No differences (p > 0.05) were observed in oxidative stress homeostasis and viability between treatments. A downregulation (p < 0.05) of CDH1 in AFP0.1 and a downregulation of AQP3 in AFP0.5 were observed in comparison to the other groups. An upregulation (p < 0.05) was detected in HSP70 and BCL2 on AFP0.5 compared to AFP0.1 group. The addition of AFP I in slow freezing solution can benefit cryopreserved sheep in vivo-derived embryos, without affecting embryonic survival.

The coasting time affects the quality of cumulus-oocyte complexes in superstimulated ewes.

We hypothesized that the coasting time may be beneficial to the quality of cumulus-oocyte complexes recovered from live ewes, as reported in cattle. The present study assessed the effect of coasting times on the quantity and quality of cumulus-oocyte complexes (COCs) in sheep. All ewes were subjected to the "Day 0 protocol", followed by an ovarian stimulation (80 mg of pFSH in three decreasing doses), varying only the coasting time [12 (G12), 36 (G36), or 60 h (G60]. In Experiment 1, data regarding follicular population was assessed. In Experiment 2, the COC quality was checked by their morphology, brilliant cresyl blue (BCB) test, evaluation of chromatin condensation pattern, and oocyte diameter. In Experiment 3, genes related to oocyte developmental competence were evaluated in BCB + COCs. The oocytes in the G60 group had more (P < 0.05) large follicles than the other groups and oocytes with a greater diameter than the G12. Oocyte morphology was similar (P > 0.05) among groups, as well as the BCB + COCs quantity. The G60-oocytes presented a better (P < 0.05) configuration of chromatin condensation compared with the other groups and a greater (P < 0.05) gene expression of BMP15, MATER, ZAR1, and PTGS2 compared with G12, and PTGS2 and HAS2 compared with G36 group. In conclusion, 60 h of coasting time positively affects the quality of COCs recovered after subjecting ewes to the "Day 0 protocol" and ovarian superstimulation. Implementing the appropriate coasting time to a given protocol can positively impact the in vitro embryo production outcomes in sheep.

Artificial Long Estrus Protocols Administered Prior to Progesterone Increase Endometrial Uterocalin Expression in Anestrous Mares.

Uterocalin (P19) is a lipocalin protein that has binding activity for the provisioning of the embryo with lipids, including those essential for morphogenesis and pattern formation. Therefore, it is thought that P19 plays an important role in supporting the survival of the early embryo. Previous research has shown that the endometrium from acyclic mares expressed more P19 following a simulated long estrus: treatment of 8 mg of estradiol benzoate (EB) split over five consecutive days, followed by long-acting progesterone administration (LA-P4) 72 hours later, when compared to a single 2.5 mg EB treatment. The main objective of this study was to evaluate if a new long estrus protocol, with fewer EB administrations, also increases endometrial uterocalin expression. Fifteen mares were assigned to three groups: long estrus 5 administrations (LE5; a total of 8 mg of EB in increasing doses was given over five consecutive days, Day -7 to Day -3); long estrus 2 administrations (LE2; 3 and 5 mg EB were given on Day -7 and Day -4); and short estrus (SE; a single dose of 2.5 mg EB was administered on Day -2). All groups received LA P4 on day 0 (D0). Daily ultrasonography and blood collections were performed to assess uterine edema and estradiol and progesterone plasma concentrations, respectively. Endometrial biopsies were collected on Day 4 to evaluate P19 expression by RT-qPCR. There were no differences in P19 expression between groups LE2 and LE5. However, P19 expression was higher (P < .05) in the LE groups than in the SE group. Both LE protocols produced high uterine edema (score 2-3) for at least 6 days. There was no effect of group (LE2 or LE5) on overall estradiol and progesterone concentrations (P > .1). In conclusion, the two EB administrations protocol is more feasible for preparing acyclic recipient mares at field conditions. However, a positive effect of the LE protocols on embryo survival and viability in anestrous treated mares still needs to be confirmed.

Addition of antifreeze protein type I or III to extenders for ram sperm cryopreservation.

Antifreeze proteins (AFP) play an important role in cellular survival at sub-zero temperatures. This study assessed the effect of AFP type I or III in semen extender (TRIS-egg yolk) for ram sperm cryopreservation. Pooled semen of four rams were allocated into five treatments: Control (CONT, without AFP); AFP Type I [0.1 (AFPI-0.1) or 0.5 (AFPI-0.5) µg/mL]; or III [0.1 (AFPIII-0.1) or 0.5 (AFPIII-0.5) µg/mL], and then frozen in six replicates. Treatments affected kinetic parameters, plasma membrane integrity and morphology (P < 0.05). The AFPIII-0.1 presented lesser total motility. Linearity was greater in AFPI-0.1, AFPI-0.5 and AFPIII-0.5 and straightness was greater in all AFP-supplemented extenders. Plasma membrane integrity was greater in AFPI-0.1 and AFPI-0.5. All AFP groups had greater percentage of normal sperm than CONT. No differences (P > 0.05) were observed in hypoosmotic test, sperm acrosome status, mitochondrial activity, chromatin condensation, perivitelline membrane binding rate and lipoperoxidation. In conclusion, the use of AFP, predominantly type I, may increase sperm cell protection during cryopreservation, with no adverse effect on potential fertilization capacity or increase in reactive oxygen species, being a potential cryoprotectant to ram sperm.

Use of oxytocin to attain cervical dilation for transcervical embryo transfer in sheep.

The aim of this work was to determine whether a cervical dilation protocol (CDP) composed of only oxytocin can be used to perform transcervical (non-surgical) embryo transfer in sheep (NSET) without affecting the viability of the corpus luteum (CL). Likewise, we evaluated whether a cervical transposing test with a Hegar dilator (CT Hegar test), performed at oestrous time, could be used to screen ewes for NSET (greater or lower chances to transpose the cervix). For that, oestrous and ovulation synchronization was performed in 25 Santa Inês ewes to induce the dioestrous condition. Animals went through the following CDP in a crossover design: E + OX, oestradiol benzoate (100 µg intravenously [IV]) and oxytocin (100 IU IV); OX, oxytocin (100 IU IV); and SAL, saline solution (IV). Using a Hegar dilator, cervical transposing attempts were performed at oestrous (D0) and dioestrous time (D8). The viability of the CL (morphology, luteal blood flow and progesterone values) was evaluated by ultrasonography (colour Doppler and B-mode) and by serum progesterone measurement from D7 to D13. The cervical transposing rate was lower for the SAL group (64%; 16/25; p < .05) and did not differ between the E + OX (88%; 22/25, p > .05) and OX (84%; 21/25, p > .05) groups. No treatment affected the CL viability. The CT Hegar test showed a high sensitivity (85.7%-93.3%), satisfactory accuracy (72%-84%), low false-negative rate (6.7%-14.6%), but high false-positive rate (46%-66.7%). In conclusion, a CDP protocol composed exclusively of oxytocin can lead to good cervical transposing rates and does not affect the viability of the CL. In addition, a screening test (CT Hegar) performed at oestrus can identify ewes for which cervical transposing will likely not occur at NSET.

Gene expression patterns of in vivo-derived sheep blastocysts is more affected by vitrification than slow freezing technique.

Transfer of fresh sheep embryos frequently results in higher pregnancy rate compared to cryopreserved ones, possibly due to a failure in the communication between the cryopreserved embryo and the endometrium during pre-implantation and pregnancy establishment. Thus, this study assessed the effect of sheep embryo cryopreservation (slow freezing or vitrification) on embryo survival rate and expression of genes related to trophectoderm differentiation (CDX2), pluripotency maintenance (NANOG), cell proliferation (TGFB1), mitochondrial activity (NRF1) and apoptosis (BAX and BCL2). Superovulation (n = 32 ewes) was performed and embryos were transcervically collected. One hundred good quality (Grade I and II) embryos were allocated into three groups: fresh embryos (CTL; n = 15), slow freezing (SF; n = 42) or vitrification (VT; n = 43). After thawing/warming, three pools of five blastocysts per group were used for RT-qPCR; the remaining 55 embryos were cultured in vitro in SOFaa medium at 38.5 °C and 5% CO2 (SF: n = 27 and VT: n = 28). Survival rate of SF and VT were, respectively, 29.6% (8/27) and 14.2% (4/28) at 24 h; and 48.1% (13/27) and 32.1% (9/28) at 48 h (P > 0.05). Only CDX2 was affected (up-regulated, P < 0.05) in both groups compared to CTL. The BAX transcript was upregulated in VT, compared to SF group. The VT increased (P < 0.05) the expression of all genes, except for NANOG and NRF1, when compared to the CTL. In conclusion, although in vitro survival was similar between techniques, VT led to increased changes in blastocyst gene expression compared to CTL and SF.

Reproductive seasonality in Saanen goats kept under tropical conditions.

This study aimed to verify the reproductive seasonality in Saanen goats from distinct parity orders (nulliparous, primiparous, and pluriparous) throughout an entire year in the Southeast Region of Brazil. In addition, it was also verified the influence of environmental indexes, metabolic profile, body weight, and social interactions on the reproductive seasonality of these animals. Forty Saanen goats kept under intensive system at latitude 22° 52' 30″ s and tropical photoperiod (11-13 h of light per day) were used. Every 15 days, blood from 24 animals (8 animals per group) was collected for assessment of serum progesterone levels. Monthly, environmental data (rainfall index, maximum and minimum temperature, and time of natural daylight) and blood were collected to evaluate the metabolic profile. Weighting and body condition score measurement were also carried out every 2 months. There was no difference in body weight and body condition score in primiparous and pluriparous goats. Nulliparous goats showed natural breeding season in the winter season while the primiparous and pluriparous goats remained cyclical in autumn and winter season. Regardless of the parity order, all goats showed a seasonal anestrus in the spring season. From December, social interactions were able to reestablish, in distinct degrees, the cyclical state in the goats. Likewise, environmental and weight indexes had low and moderate influence, respectively, on the reproductive seasonality in the Saanen goats kept under tropical conditions.

Supplementation of 17ß-estradiol and progesterone in the co-culture medium of bovine oviductal epithelial cells and ovine spermatozoa reduces the sperm kinematics and capacitation.

This study investigated the effect that bovine oviductal epithelial cell (BOEC) and ovine spermatozoa co-culture exposed to different hormonal environments had on ram sperm function over the course of a 24-h incubation period. Ram cooled-stored spermatozoa were selected by swim-up and then co-cultured separately for 24 h at 38.5 °C under 5% CO2 with either: (1) Fert-TALP medium (positive control [POSControl]), (2) Fert-TALP medium supplemented with 17ß-estradiol (E2) and progesterone (P4) at concentrations similar to follicular phase (Follicular NEGControl), (3) Fert-TALP medium supplemented with E2 and P4 concentrations similar to luteal phase (Luteal NEGControl), (4) BOEC cultured in the same medium as that of the Follicular NEGControl group (Follicular BOEC group), or (5) BOEC cultured in the same medium as that of the Luteal NEGControl group (Luteal BOEC group). The sperm kinematics, capacitation status, and plasma membrane (PM) integrity were evaluated at different intervals. Sperm PM integrity was not affected (P ˃ 0.05) by BOEC co-culture, regardless of the phase of the estrous cycle. After 4 h of incubation, the Luteal BOEC group presented lower (P < 0.05) progressive motility and total motility than the Luteal NEGControl group while the Follicular BOEC group showed lower (P < 0.05) velocimetric parameters and progressive motility than the Follicular NEGControl group. Throughout the incubation period, both BOEC co-culture groups showed a decrease (P < 0.05) in their capacitation rate in comparison to the POSControl group. Conversely, the Luteal BOEC group presented a higher (P < 0.05) non-capacitated rate than both the POSControl and Luteal NEGControl groups. In conclusion, BOEC co-culture with ovine spermatozoa at either the follicular or luteal phase decreases sperm kinematics and delays sperm capacitation.

Dose and administration protocol for FSH used for ovarian stimulation affect gene expression in sheep cumulus-oocyte complexes.

The present study evaluated the effect of four ovarian stimulation protocols on the follicular population and molecular status of cumulus-oocyte complexes (COCs). Twelve Santa Inês ewes (in a cross-over design) received 80 or 120mg FSH alone in a multiple-dose (MD80 and MD120) regimen or in combination with 300IU equine chorionic gonadotrophin (eCG) in a one-shot (OS80 and OS120) protocol. The follicular population, COC recovery rate, mean COCs per ewe and the rate of brilliant Cresyl blue-positive (BCB+) COCs were similar among treatments (P>0.05). The expression of markers of oocyte competence (ZAR1, zygote arrest 1; MATER, maternal antigen that embryo requires; GDF9, growth differentiation factor 9; BMP15, bone morphogenetic protein 15; Bcl-2, B-cell lymphoma 2; BAX, Bcl-2 associated X protein) and the steroidogenic pathway (ERα, oestrogen receptor α; LHr, LH receptor; FSHr, FSH receptor; STAR, steroidogenic acute regulatory protein) was affected by stimulation. Specifically, the expression of markers of the steroidogenic pathway was reduced with increasing FSH dose in the OS protocol. FSH at a dose of 80mg reduced the expression of FSHr and ERα in the OS versus MD protocol. Conversely, in MD protocol, only LHr was affected by increasing FSH dose. In conclusion, 80mg FSH in the MD or OS protocol was sufficient to promote the development of multiple follicles and obtain fully grown (BCB+) oocytes. The MD protocol may be more appropriate for the production of better-quality oocytes.

l-carnitine supplementation during vitrification or warming of in vivo-produced ovine embryos does not affect embryonic survival rates, but alters CrAT and PRDX1 expression.

l-carnitine is an antioxidant and ß-oxidation stimulator substance commonly used to improve metabolic performance of oocytes and embryos in in vitro systems. However, few studies have evaluated its beneficial effects in embryos produced in vivo. This study aimed to evaluate the effect of l-carnitine supplementation into vitrification or warming solutions on the post-warming character of day 6-7 in vivo-produced ovine embryos. l-carnitine (3.72 mM) was added to vitrification (Experiment 1) or warming solutions (Experiment 2). In experiments 1 and 2, the embryos were vitrified using straw and cryo-tip protocols, respectively. In vitro culture (IVC) of warmed embryos was performed for 72 h in order to evaluate survival rates, reactive oxygen species (ROS) levels, total cell number (TCN), number of apoptotic cells, apoptotic index evaluation, and gene expression analysis of carnitine palmitoyltransferase I and 2 (CPT1 and CPT2), carnitine O-acetyltransferase (CrAT), and peroxiredoxin-1 (PRDX1). In experiment 1, survival rate, ROS levels after 24 h of IVC, total cell number at 24 h and 72 h, apoptotic cells and apoptotic index at 72 h of IVC were similar in embryos vitrified in medium supplemented with LC or not. Gene expression analysis showed no differences in CPT1 and CPT2 mRNA relative abundance in embryos of both experiments compared to fresh embryos (FE); however, CrAT was downregulated (p < 0.05) in C1, and PRDX1 was downregulated (p < 0.05) in both the control (C1) and l-carnitine (LC1) groups, compared to FE. Moreover, CrAT and PRDX1 were upregulated (p < 0.05) in C2, and CrAT was downregulated (p < 0.05) in LC2, in relation to FE. Although the short-term LC supplementation at 3.72 mM did not improve survival, and quality parameters of in vivo-produced ovine embryos, it could affect their quality at a molecular level. In conclusion, further investigations with different concentrations of LC and tools are needed for improvement of the efficiency of these strategies.

Non-surgical embryo transfer in goats and sheep: the Brazilian experience.

Brazil has presented tremendous progress in non-surgical embryo transfer (NSET) in sheep and goats. New instruments and techniques for non-surgical embryo recovery (NSER) and NSET in small ruminants were implemented. Recent improvements include refinement of the protocols for cervical relaxation combining oestradiol-oxytocin-cloprostenol treatment at specific times before NSER in sheep; recipient goats do not require any hormonal drugs to induce cervical dilation and direct embryo transfer by the cervical route yields excellent results. Transrectal ovarian ultrasonography (B-mode but especially colour Doppler) have proven to be accurate methods to localise and enumerate corpora lutea and luteinised unovulated follicles in recipient and donor does and ewes. An array of new criteria for selecting superior animals for NSER and NSET (e.g. cervical mapping) have been developed by Brazilian researchers. Extensive studies on both technologies were initially conducted in commercial breeds of goats and sheep but have been gradually extended to some native breeds of sheep (germplasm conservation) and dairy goat operations. It is speculated that, in future, NSER and NSET may become methods of choice for caprine and ovine embryo recovery and transfer in Brazil, and then globally. Due primarily to the efficiency of NSET in goats, a novel interspecies (e.g. bovine) IVP method may soon be developed on a large scale. The Brazilian experience is an invaluable source of information and know-how promoting the replacement of conventional surgical assisted reproductive technologies with non-surgical procedures and hence supporting the rapid development of the embryo transfer industry in small ruminants.

Evaluation of cervical mucus and reproductive efficiency of seasonally anovular dairy goats after short-term progestagen-based estrous induction protocols with different gonadotropins.

The use of three different gonadotropins was tested for estrous induction in dairy goats during the non-breeding season. All does received an injection of 30 µg of d-cloprostenol and intravaginal sponges containing 60mg of medroxyprogesterone acetate (MAP) for 6 d plus 20 IU of porcine FSH (pFSH), 200 IU of eCG or 250 IU of hCG 24h before sponge removal. In Experiment 1 (n=24), ovarian ultrasound parameters were recorded and cervical mucus was evaluated daily for 5 d after sponge removal or until ovulation. In Experiment 2 (n=80), reproductive efficiency of artificially inseminated or naturally mated does was assessed. The mean interval from sponge removal to ovulation (73.5±23.7 h), number of ovulations (1.6±0.7) and ovulatory follicle diameter (7.2±0.8 mm) did not vary (P >0.05) among the three groups. At ovulation, cervical mucus had crystalline-striated to striated (22.2%), striated to striated-caseous (72.2%) and striated-caseous to caseous (5.6%) appearance. The largest follicle diameter was greater (P <0.05) in does with crystalline (6.7±1.4 mm), crystalline-striated (7.2±1.1 mm) or striated (7.3±1.3 mm) mucus than in those with striated-caseous (5.3±1.4 mm) or caseous (4.5±1.1 mm) mucus. Percentage of animals exhibiting estrus (92.5%) and conception rate (60.8%) were similar (P >0.05) among the three gonadotropins groups. Results of this study support the use of eCG (200 IU), hCG (250 IU) and pFSH (20 IU) for the estrous induction protocols in dairy goats during the non-breeding season. Cervical mucus evaluation can be used as an additional method to determine the optimal time for artificial insemination in goats.

Vascularização uterina de éguas prenhes de jumento e de garanhão monitoradas por ultrassonografia Doppler / Uterine vasculature of pregnant mares of donkey and stallion through the Doppler ultrasonography spectral mode

Este estudo teve como objetivo analisar e descrever os achados ultrassonográficos do útero de éguas gestantes de diferentes idades, de jumento e garanhão, utilizando a técnica de ultrassonografia Doppler Espectral, visando caracterizar os índices de resistência (RI) e pulsatilidade (PI) até 154 dias de gestação. Foram utilizadas 20 éguas em idade reprodutiva, sem raça definida. As avaliações foram realizadas nas artérias uterinas. Não foi constatada interação entre os valores de RI e PI obtidos nos cornos uterinos (que iniciaram ou não a gestação) com o tempo da gestação (P>0,05), desta forma os dados foram discutidos separadamente. Não foram encontradas diferenças entre as avaliações de RI e PI quando comparado os cornos que iniciaram ou não a gestação (P>0,05). Verificou-se efeito de tempo (P<0,05) sobre o PI, onde no D70 foi constatada a menor média quando comparado aos tempos D0, D7, D14 e D21. Não f oi encontrado efeito de tempo sobre o RI (P>0,05). As éguas gestantes de jumento apresentaram maior vascularização uterina no corno não gestante, que as éguas gestantes do garanhão. Com exceção do RI no lado não gravídico, foi verificado efeito de idade da fêmea sobre o lado da gestação (P<0,05), onde as éguas idosas, tanto no corno que iniciou a gestação e o contralateral, tiveram PI e RI mais altos, ou seja, menos vascularizados. Concluiu-se que a vascularização uterina não apresentou diferença entre os cornos uterinos. Há um aumento da vascularização uterina no 70º dia de gestação. Éguas gestantes de jumentos apresentaram maior vascularização uterina, quando comparada às éguas gestantes de garanhões. Éguas com idade acima de 15 anos apresentaram menor vascularização uterina, quando comparadas com idade inferior a 15 anos.(AU)

This study aimed to evaluate and describe the ultrasonographic findings of uterus of pregnant mares of different ages, mated by either ass or stallion, using the spectral Doppler ultrasonography, aiming to characterize the resistance (RI) and pulsatility (PI) Doppler indices up to 154 days of pregnancy. A total of 20 mares in reproductive age of non-defined breed were used. The evaluations were performed in uterine arteries. There was no interaction between RI and PI values obtained in uterine horns (that initiated or not the pregnancy) with time of pregnancy (P>0.05), thus, data were discussed separately. There were no differences between RI and PI evaluations when compared to the uterine horns that initiated or not the pregnancy (P>0.05). A time effect was observed (P<0.05) on PI, when D70 presented lower averages than D0, D7, D14 and D21. There was no time effect (P>0.05) on RI. Pregnant mares mated by asses presented higher uterine vascularization in non-pregnant horn than pregnant mares mated by stallions. With the exception of RI in non-pregnant horn, there was a female age effect on the side of pregnancy (P<0.05), where aged mares in both - pregnant and non-pregnant horns - showed higher PI and RI, so less vascularized. It is possible to conclude that the uterine vascularization did not present any difference between uterine horns. There is an increase in the uterine vascularization on the 70th day of pregnancy. Pregnant mares mated by assess presented higher uterine vascularization when compared to those mated by stallions. Mares aging over 15 years old presented lower uterine vascularization, when compared to those younger.(AU)

Luteal dynamic and functionality assessment in dairy goats by luteal blood flow, luteal biometry, and hormonal assay.

This study aimed to assess the luteal dynamics of pregnant and non-pregnant Saanen goats throughout an estrous cycle by B-mode and color Doppler ultrasonography (US) associated with a P4 hormonal assay. Furthermore, a cutoff point was chosen to determine the corpus luteum (CL) functionality by luteal biometry (LB) measurement and luteal blood flow (LBF) assessment. Ultrasound assessment was carried out daily throughout an entire estrous cycle (21 days) in 23 Saanen goats pre-synchronized and inseminated in the breeding season. The plasmatic P4 concentration was determined daily by radioimmunoassay. LB parameters (diameter, area, and volume) were measured using the maximum area of a cross-section of the CL. LBF assessment was performed subjectively by percentage of area of colored pixels and objectively by calculating the number of the colored pixels. Eventually, 45.0% (9/20) and 55.0% (11/20) of goats became pregnant and or remained non-pregnant, respectively. The LB and LBF demonstrated value stabilization on the 9th day of the estrous cycle and maximum values on the 12th and 13th days of the estrous cycle, respectively. LB presented a progressive decrease in the luteal regression phase, whereas the LBF decreased abruptly in association with P4. The LBF values were more reliable in predicting the luteal functionality when compared to the LB data. The number of colored pixels accurately predicted values of P4 >1.0 ng/mL, reaching only 17% of the maximum values, and 1200 colored pixels as a minimum cutoff point when compared to the use of 53% of the maximum values and a minimum luteal diameter of 9.0 mm as cutoff point for P4 >1.0 ng/mL. The LBF assessment was more informative about the CL functionality throughout the complete luteal phase when compared to the LB. The use of the number of colored pixels is indicated for research regarding luteal functionality due to their greater correlation with P4 values. In addition, the luteal subjective evaluation can be used under field conditions due to greater convenience and similar pattern of correlation with P4.

Nonsurgical embryo recovery and transfer in sheep and goats.

The embryo transfer techniques used in small ruminants worldwide are based in surgical procedures. These actions are performed under general anesthesia which needs a combination of animal fasting and drugs for secure animal handling and surgery manipulations. Therefore, it involves risks to animal health and life. The major limiting sequels are adhesions formed by the abdominal surgery, in the ovaries, uterus, or between them. These occurrences can both compromise uterus accessing and oocyte capture and are responsible for decreasing success and limiting successive embryo collections. In contrast, nonsurgical embryo procedures can be performed in a relatively simplified way. Nonsurgical embryo recovery does not need animal prolonged starvation, drug retention is minimized, and donors can stay in a standing position. After the end of embryo recovery, donors are promptly restored to their routine housing and feeding. Furthermore, this technique does not need incisions and, therefore, can be used repetitively in superovulated or nonsuperovulated goats and sheep for embryo recovery-a similar procedure done in cattle. In Brazil, promising results are reported using nonsurgical embryo transfer in recipient goats, and studies are currently evaluating similar procedures in sheep. Therefore, this review aimed to present the current panorama of nonsurgical embryo transfer in sheep and goats.

Presence of leptospires on genital tract of mares with reproductive problems.

Leptospirosis is a zoonotic disease of global importance, and has a worldwide distribution. Equine leptospirosis is commonly manifested by recurrent uveitis, reproductive disorders, as abortions, embryonic absorption, stillbirth and the birth of weak foals. The aim of this study was to verify the presence of Leptospira sp or its DNA in genital tract of mares with reproductive problems. A total of 38 mares with reproductive problems were studied. All the mares were sampled for blood (for serology), urine (for culturing and qPCR), vaginal fluid-VF and endometrial biopsy-EB (for culturing, qPCR and indirect immunofluorescence). PCRs products were sequenced for secY gene. Seventeen (44.7%) serum samples were reactive, predominantly against serogroups Australis (76.4%) and Pomona (23.6%). No positive culture was obtained, but DNA was detected by qPCR on urine samples (26.3%), VF (44.7%) and EB (18.4%) collected 2 months or longer following diagnosis of early fetal death and endometritis. Leptospira cell aggregations were visible by indirect immunofluorescence on 57.1% (4/7) EBs and 17.6% (3/17) VFs. A total of 18 amplicons showed interpretable sequences. Out of those 18 amplicons, 15 presented 100% of identity with the species L. interrogans (sv Bratislava and Pomona), while three were L. borgpertersenii. This study suggests the presence of leptospires in the uterus of mares with reproductive problems. Moreover, serology was shown not to be indicated for the diagnosis of presumptive Leptospira infection in early gestation. The most common agent of the genital infection in those mares was L. interrogans, most probably sg Australis.

Progestin-impregnated intravaginal sponges for estrus induction and synchronization influences on goats vaginal flora and antimicrobial susceptibility.

The objective was to characterize vaginal bacteria, their antimicrobial sensitivity, and the incidence of vaginitis, in goats before and after insertion of intravaginal sponges containing progesterone. Sponges were inserted in 37 Saanen goats and removed after 6, 9 or 12d (G6, G9 and G12). At sponge removal, all goats had clinical signs of vaginitis. Sampling was conducted just before sponge insertion and at 0, 24, 48, and 72 h after sponge removal. Vaginal secretions were subjected to standard bacteriological procedures, including isolation of bacteria, subculture, and determination of sensitivity to antimicrobials (gentamicin, cefalotin, tetracycline, ciprofloxacin, trimethoprim-sulfamethoxazole, amoxicillin and clavulanic acid, penicillin G and cefoxitin). Ciprofloxacin, gentamicin, tetracycline and trimethoprim-sulfamethoxazole were the most effective for coliforms (100% sensitivity), whereas ciprofloxacin, gentamicin and tetracycline were the most effective for cocci (100, 98.6 and 97.2% sensitivity, respectively). In contrast, the least effective antimicrobials were cefalotin for the coliforms, and penicillin for the cocci (37.5 and 64.4% sensitivity, respectively), regardless of duration of implant presence and interval from implant removal to sampling. In conclusion, insertion of intravaginal progestin-impregnated sponges induced clinical vaginitis in goats. Members of Staphylococcus genus were the most frequently recovered species of the vaginal samples cultured, and all isolates were resistant to several antimicrobials.