ABSTRACT The objective of this study was to evaluate the relationships between preovulatory follicle (POF) and corpus luteum (CL) diameters, and POF and CL vascular perfusion with progesterone production, ovulation, and pregnancy in Nellore cows subjected to timed artificial insemination (TAI). Nellore cows (n = 201) were subjected to ovulation synchronization and later to ultrasound evaluation of POF and CL at the time of insemination (D0) and seven days later (D7), respectively. Females were divided into three categories according to the POF diameter assessed at the time of insemination: small (SF), medium (MF), and large (LF) follicles. The LF group had a greater number and intensity of pixels in the POF ultrasound exam compared with the SF group. The CL flow intensity and progesterone concentration were also higher in the LF group. The SF group showed lower flow intensity and lower ovulation rate compared with the others. When non-pregnant females were compared to pregnant ones, no difference was observed in any of the analyzed variables. The results show for the first time in Nellore cattle the relationship between the size of ovarian structures and blood flow (quantity and intensity) as well as the ability of the CL to produce progesterone. The intensity of the POF pixels proved to be relevant, demonstrating correlations with the size and flow of the CL, which were not found when evaluating only the number of pixels, thus revealing the importance of evaluating complementary characteristics of the flow. (POF (CL diameters production TAI. TAI . (TAI) n 201 D0 D (D0 D7, D7 , (D7) respectively SF, (SF) MF, MF (MF) (LF follicles others nonpregnant non ones variables quantity relevant (TAI 20 (D (D7 (SF (MF 2

Resumo O parabasalídeo Pentatrichomonas hominis é geralmente considerado um componente simbiótico da microbiota gastrointestinal em uma ampla variedade de hospedeiros vertebrados, incluindo os humanos. No entanto, um número limitado de estudos levantou a possibilidade de que possa atuar como patógeno de humanos, cães e suínos, e que algumas infecções humanas possam ter origem zoonótica. Dados da América do Norte revelaram uma associação entre P. hominis e o trato urogenital bovino, principalmente, em touros e raramente em vacas. A importância dessa observação está ligada a uma possível interferência no diagnóstico preciso do patógeno venéreo de importância econômica Tritrichomonas foetus. O presente estudo empregou métodos baseados em cultura em combinação com técnicas moleculares, para examinar as cavidades prepuciais de quatro touros, criados em pastagem aberta no sudeste do Brasil, quanto à presença de tricomonas. Protozoários móveis foram isolados de três dos touros e foram definitivamente identificados como P. hominis com base no sequenciamento de nucleotídeos de amplicons geradas pela reação em cadeia da polimerase (PCR), empregando a região RNA ribossômico (ITS1-5.8S rDNA-ITS2) do parasita como alvo. Os potenciais implicações desses achados para a saúde bovina e humana são discutidas.

Abstract The parabasalid Pentatrichomonas hominis is generally considered to represent a symbiotic component of the gastrointestinal microbiota in a wide variety of vertebrate hosts including humans. Nevertheless, a limited number of studies have raised the possibility that it may act as a pathogen of humans, dogs, and pigs and that some human infections may have a zoonotic origin. Data from North America revealed an association between P. hominis and the bovine urogenital tract, principally in bulls and rarely in cows. The importance of this observation is linked to possible interference in the accurate diagnosis of the economically important venereal pathogen Tritrichomonas foetus. The current study employed culture-based and molecular methods to examine the preputial cavities of four breeding bulls, raised in open pasture in southeastern Brazil, for the presence of trichomonads. Motile protozoa were isolated from three of the bulls and were definitively identified as P. hominis based on nucleotide sequencing of polymerase chain reaction (PCR) amplicons derived from the ribosomal RNA operon (ITS1-5.8S rDNA-ITS2) of the parasite. The potential implications of these findings for bovine and human health are discussed.

ABSTRACT The objective of this study was to evaluate the effect of timed artificial insemination (TAI) on pregnancy rates, reproductive efficiency, and mean weaning weight of calves at the end of breeding season in Nellore females. In this work, 744 Nellore females were divided into two groups: in group I (n = 396), the females were subjected to natural service during the breeding season, which lasts for 180 days. In group II (n = 348), a hormonal protocol was performed for synchronization of ovulation, and the females were subjected to TAI. Afterwards, these latter females were then serviced by a cleanup bull until the end of the breeding season. The group II obtained a higher pregnancy rate as well as higher reproductive efficiency compared with group I. The weaning weight of group II calves was greater than that of group I. The use of TAI combined with cleanup bulls enables higher pregnancy rates and a better reproductive efficiency in relation to the use of only natural service. In addition, weaning calves born from TAI are heavier than calves born from natural service.

ABSTRACT The present study aimed to correlate the age and weight of Girolando heifers with uterine and ovarian development. Sixty heifers between 12 and 36 months old were weighed and subjected to ultrasound assessment to measure the diameters of each uterine horn and ovaries, monthly. These measures continued until their first ovulation. The animals were divided, for data analysis, according to their age into five groups: GI (12 to 14 months), GII (15 to 18 months), GIII (19 to 24 months), GIV (25 to 30 months), and GV (31 to 36 months). The dispersion diagram was used to assess the correlation between data collected monthly from uterus/ovaries and weight/age. An average daily growth of 0.0032 mm was recorded for the right ovary (RO) and 0.0051 mm for the left ovary (LO). The average size of the RO in GI was 17.58 mm and the LO was 15.28 mm; in GII, the RO was 16.72 mm and the LO was 15.89 mm; in GIII, the RO was 18.37 mm and the LO was 16.55 mm; in GIV, the RO was 19.09 mm and the LO was 17.49 mm; and in GV, the RO was 19.89 mm and the LO was 18.96 mm. The uterine horns showed an average daily growth of 0.0064 mm and 0.0071 mm for the right (RU) and left (LU) uterine horn, respectively. The average sizes of the uterine horns per group were: RU, 11.46 mm and LU, 11.48 mm; RU, 12.41 mm and LU, 12.63 mm; RU, 13.16 mm and LU, 13.19 mm; RU 14.30 mm and LU, 14.58 mm; and RU, 16.12 mm and LU, 16.68 mm for the groups GI, GII, GIII, GIV, and GV, respectively. The heifers showed an average daily weight gain of 0.224 kg and the groups GI, GII, GIII, GIV, and GV had an average weight of 220.5, 239.3, 287.6, 332.9, and 380.1 kg, respectively. Therefore, weight growth correlates with the growth of the ovaries and uterine horns; furthermore, these data correlate positively as the animals age.

ABSTRACT In this study, we investigated the effects of eCG and GnRH on the induction of puberty in prepubertal gilts and evaluated the pregnancy rate and litter size in these females. Thirty 140-day-old gilts with an average weight of 80 kg were randomly divided into two treatments: hormonal treatment (n = 15): 1000 IU of eCG and 50 μg of GnRH; and control group (n = 15): induction of estrus by male presence. Gilts were inseminated at the second estrus and pregnancy was confirmed by the non-return to estrus. At farrowing, litter size, average piglet weight, and number of stillborn and mummified piglets were recorded for analysis. The variable pregnancy rate was subjected to the Chi-square test (χ2), while variables age at first estrus, weight at insemination, and duration of first and second estrus, litter size, average weight, and average number of dead and mummified piglets were analyzed by the t test. Hormonal treatment induced puberty in 100% of the gilts in four days. There was a difference in age at first estrus and in weight at insemination. Animals in the control group had the first estrus later and weighed more than those under hormonal treatment (167 vs. 143 days, 117 vs. 102 kg, respectively). However, there was no difference between the two groups in the duration of first and second estrus (54 vs. 49 h, 45 vs. 36 h, respectively), in pregnancy rate (100 vs. 86%, respectively), litter size, average piglet weight, and number of dead and mummified piglets between the hormonal treatment and control animals (9.0 vs. 9.8 piglets, 1.3 vs. 1.3 kg, and 3.0 vs. 2.3 dead and mummified, respectively). The hormonal treatment is effective in inducing precocious puberty without impairing pregnancy rate and litter size as well as average weight of piglets.

Resumo: O objetivo deste trabalho foi avaliar os efeitos da doadora, estação do ano, do touro e do tipo de sêmen, na produção in vitro de embriões da raça Sindi (Bos indicus). Para isso, avaliaram-se 434 sessões de aspiração folicular in vivo (OPU), realizadas em 152 doadoras, com a utilização de sêmen sexado e não sexado de 22 touros. Analisaram-se as seguintes variáveis: doadora; idade da doadora; estação do ano; touro; tipo de sêmen; oócitos recuperados; viáveis e degenerados; e as taxas de clivagem e de blastocistos. Observaram-se efeitos da doadora e do touro sobre os oócitos recuperados, viáveis e degenerados e sobre a taxa de blastocisto. O ano foi dividido em estações chuvosa, de outubro a março, e seca, de abril a setembro. Na estação chuvosa, a proporção de oócitos viáveis aumentou, enquanto a de degenerados diminuiu. As doadoras com menos de seis anos tiveram maior proporção de oócitos viáveis e menor, de degenerados. O sêmen não sexado obteve as melhores taxas de clivagem e de blastocistos. Doadoras de até seis anos apresentam a maior proporção de oócitos viáveis, e o maior número de oócitos viáveis é produzido na estação chuvosa. O sêmen não sexado resulta em melhores taxas de clivagem e de blastocistos.

Abstract: The objective of this work was to evaluate the effects of donor, season of the year, sire, and type of semen on in vitro embryo production of the Sindhi breed (Bos indicus). To this end, 434 in vivo ovum pick up (OPU) sessions, performed in 152 oocyte donors, were evaluated using sex-sorted or unsorted semen from 22 sires. The following variables were analyzed: donor; age of the donor; season of the year; sire; type of semen; recovered, viable, and degenerated oocytes; and cleavage and blastocyst rates. The effects of the donor and sire were observed on recovered, viable, and degenerated oocytes, and on blastocyst rates. The year was split into rainy season, from October to March, and dry season, from April to September. In the rainy season, the proportion of viable oocytes increased, while the proportion of the degenerated ones reduced. Donors with less than six years of age had a higher proportion of viable oocytes and a lower proportion of degenerated ones. Unsorted semen obtained the best cleavage and blastocyst rates. Donors with up to six years of age show the greatest proportion of viable oocytes, and the greatest number of viable oocytes is produced during the rainy season. Unsorted semen shows the best rates for cleavage and blastocysts.

The objective of this study was to evaluate the effect of the follicular wave synchronization method on the superovulatory response of Girolando embryo donors. Seven cows and five heifers were divided into three treatments according to the method of the follicular wave synchronization prior to superovulation. The donors were superovulated with eight FSH decreasing doses, adding 133 mg per animal. The superovulatory response was evaluated using an ultrasound device to quantify the number of corpora lutea. The number and quality of recovered structures were also evaluated. Animal category (cow or heifer) and genetic group influenced the superovulatory response, in which heifers showed a greater response (100% in all treatments) compared with cows, which showed 85.7%, 57.1% and 57.1% superovulatory response for standing estrus, GnRH and P4 + BE groups, respectively. The genetic group also showed influence on superovulatory response, in which the 3/8 animals had an average of 10.3 corpora lutea per donor compared with the 3/4 animals, which showed 4.9. The follicular wave synchronization treatments did not show differences in superovulatory response, recovered structures and viable structures. The treatment of follicular wave synchronization with GnRH or with P4 + BE can be used in Girolando donors with the same efficiency as the standing estrus group, and 3/8 Girolando heifers respond better to the superovulatory treatment.

The objective of this study was to evaluate the effects of egg yolk and seminal plasma on the viability of cryopreserved goat semen. To this end, four fertile Saanen bucks, aged between 10 months and 1 year, and weighing 18 to 25 kg, were used. Semen was collected from each buck by the artificial vagina method at the end of breeding season (June-July). The extender used was the yolk citrate, which was split into two equal aliquots: 5% egg yolk (2.5 mL egg yolk: 47.5 mL citrate solution) were added to one of the samples and 10% egg yolk (5.0 mL egg yolk: 45.0 mL citrate solution) were added to another. The sperm motility and vigor after thawing and post thermal resistance test (TRT) were evaluated and the data were subjected to analysis of variance and means were compared by the F test at 5.0% probability. The observed values for motility and vigor after thawing and post thermal resistance test (TRT), fast and slow, according to the presence of seminal plasma and egg yolk percentage were: 5% egg yolk with plasma (25.0% and 3.3; 1.60% and 0.7; 12.36% and 1.6, respectively); 5% egg yolk without plasma (23.61% and 3.1; 1.25% and 0.2; 9.93% and 1.3, respectively); 10% egg yolk with plasma (30.8% and 3.3; 4.4% and 1.9; 19.5% and 2.7, respectively); and 10% egg yolk without plasma (13.4% and 2.5; 4.1% and 0.5; 17.0% and 1.0, respectively). There were significant differences between the analyzed data in relation to semen with or without plasma at different percentages of egg yolk, and the group that presented the best results was 10% egg yolk citrate in extender with plasma. The presence of seminal plasma and higher concentration of egg yolk in extender provide a higher viability of cryopreserved goat semen.

The objective of this study was to evaluate the reproductive parameters of Sindhi cows aiming to improve the efficiency of fixed-time artificial insemination protocols in this breed. For this purpose, sixteen Sindhi cows were divided into two groups: in group I (EB, n = 8) animals received an intravaginal P4 implant and an application of 2 mg of estradiol benzoate on day 0. On day 8 the implant was removed and 10 mg of Dinoprost were applied; on day 9, 1 mg of estradiol benzoate was applied, and animals were fixed-time artificially inseminated 36 hours later; in group II (EB + eCG, n = 8) animals were subjected to same treatment, and on day eight 400 IU of eCG were applied. From day 0 animals were examined daily by a trans-rectal ultrasound device, where in each assessment the largest follicles of each day were measured. The reproductive parameters observed for groups I and II were: emergence of follicular wave, 4.5±1.4 and 3.8±0.3 days; interval between wave emergence and ovulation, 5.8±1.4 and 7.1±0.3 days; interval between wave emergence and implant removal, 3.5±1.4 and 4.1±0.3 days; interval between implant removal and ovulation, 74.0±11.8 and 82.5±4.2 hours; diameter of the dominant follicle (DF) at the time of implant removal, 7.5±2.5 and 6.6±1.5 mm; maximum diameter of DF, 10.47±8.85 and 3.7±1.2 mm; growth rate of DF, 0.90±0.83 and 0.7±0.6 mm/day; ovulation rate, 75% and 100%; and pregnancy rate, 0% and 50%, respectively. There was no significant difference between the groups. We observed that eCG was not efficient in improving the reproductive parameters analyzed in this breed.

O objetivo deste estudo foi avaliar os protocolos de capacitação espermática in vitro com 100 mg/ml de heparina e 5 mM de cálcio ionóforo e correlacionar a capacitação com a fertilidade in vivo. A capacitação espermática foi avaliada pela coloração com iodeto de propídio e diacetato de carboxifluoresceína e pela coloração tripla com vermelho congo, vermelho neutro e giemsa. Os espermatozóides foram avaliados quanto a viabilidade (vivos ou mortos) e a qualidade do acrossomo (lesados ou íntegros), sendo caracterizados como capacitados (vivos e lesados), não capacitados (vivos e íntegros) e mortos (lesados ou íntegros). A heparina apresentou 64,54% e 39,16% e o cálcio ionóforo 36,41% e 18,11% de espermatozóides capacitados, respectivamente, pela epifluorescência e coloração tripla. Os touros foram divididos em três grupos de fertilidade, sendo o Grupo A <63%, o Grupo B entre 63 e 68% e o Grupo C >68%. Nos três grupos houve diferença significativa (p<0,01) em relação aos espermatozóides capacitados dos tratamentos com heparina e cálcio ionóforo, tanto na epifluorescência quanto na coloração tripla. Não houve diferença estatística significativa (p>0,01) para os espermatozóides capacitados, não capacitados e mortos entre os grupos A, B e C, tanto na epifluorescência quanto na coloração tripla para ambos capacitores (heparina e cálcio ionóforo). Não houve correlação entre os espermatozóides capacitados in vitro e a taxa de fertilidade a campo. A heparina apresentou melhor taxa de espermatozóides capacitados e a epifluorescência mostrou-se mais eficiente na detecção da capacitação espermática (p<0,01).

The aim of this particular study was to test in vitro sperm capacitation protocols, using heparin (100mg/ml) and calcium ionophore (5mM). Propidium iodide and carboxifluorescein diacetate (IP/CFDA) in a fluorescence microscope as well as triple stain (congo red, neutral red and Giemsa) in Phase contrast microscope were used as staining. The spermatozoa were classified according to its viability (alive or dead) and acrossome integrity (damaged or intact). They were considered as follows: capacitated (alive and damaged); non capacitated (alive and not damaged) and dead (damaged or intact). The heparin group showed a ratio of 64.54% and 39.16% of capacitated spermatozoa in IP/CFDA and triple stain, respectively. In the calcium group, 36.41% and 18.11% of spermatozoa were capacitated in IP/CFDA and triple stain, respectively. Bulls were divided into 3 groups according to their fertility rates as follows: Group A < 63%, Group B from 63 to 68% and Group C > 68%. For all three groups there was significant differences (p<0.01), regarding capacitated spermatozoa with heparin and calcium ionophore in both stains IP/CFDA and triple stain. On the other hand, there was no significant differences (p>0.01), when observed capacitated, non capacitated and dead spermatozoa among groups A and B; A and C; B and C, using heparin and calcium ionophore in both stains. No correlation was seen between capacitation and fertility rates. Therefore heparin treatment showed better sperm capacitation rates than calcium ionophore. The IP/CFDA technique showed itself as being a better method to evaluate sperm capacitation than the triple stain (p<0.01).

Este trabalho avaliou o desenvolvimento in vitro e in vivo de mórulas de camundongos congeladas por diferentes métodos. A congelação lenta foi realizada em 1,5M de etileno glicol (EG) sendo os embriões resfriados a 0,5; 0,7; 1,0 e 1,2ºC/minuto. Na vitrificação, as mórulas foram equilibradas por 3 minutos em 20% de EG e vitrificadas em solução contendo 40% de EG, 18% de ficol e 10,26% de sacarose após 60 segundos de exposição. A congelação rápida em vapor de nitrogênio foi realizada em solução contendo 3M de EG + 0,3M de sacarose após 2 ou 5 minutos de exposição. Os embriões dos três métodos foram descongelados pela exposição das palhetas ao ar por 10 segundos e imersão em água a 25ºC por 20 segundos. Todos os embriões descongelados foram cultivados in vitro por 72 horas para avaliação da sobrevivência in vitro. Para avaliação da sobrevivência in vivo, mórulas congeladas e não congeladas (controle) foram transferidas para receptoras. O desenvolvimento in vitro nas velocidades de 0,5; 0,7; 1,0 e 1,2ºC/minuto foi, respectivamente, 72,3; 79,6; 76,5 e 84,8%. Não houve diferença estatística entre as velocidades de 0,7; 1,0 e 1,2ºC/minuto (p > 0,01). O desenvolvimento in vitro das mórulas congeladas por vitrificação e pelo método rápido (84,5 e 74,3%, respectivamente) foi semelhante ao método lento. In vivo, a taxa de implantação e o número de fetos vivos não diferiram estatisticamente entre os grupos lento a 1,2ºC/minute, vitrificação e rápido.

The in vitro and in vivo development of mouse morulae after cryopreservation through different methods was examined. The slow-freezing involved an equilibration in 1.5M ethylene glycol (EG) and cooled at 0.5; 0.7; 1.0 or 1.2ºC/minute. The vitrification involved a 3 minutes equilibration in 20% EG and 60 seconds in solution containing 40% EG, 18% ficoll and 10.26% sucrose. The quick-freezing involved an equilibration in 3M EG + 0.3M sucrose for 5 minutes and 2 minutes in nitrogen vapor. In all three methods the straws were thawed in air for 10 seconds and in water at 25ºC for 20 seconds and the embryos cultured in vitro for 72 hours to estimate blastocyst rate. To assess viability in vivo, frozen morulae as well as fresh embryos were transferred into recipients. The in vitro development rates with 0.5, 0.7; 1.0 and 1.2ºC/minute were, respectively, 72.3; 79.6; 76.5 and 84.8%. There was no significant difference among the cooling rates of 0.7; 1.0 and 1.2ºC/minute (p > 0.01). The in vitro survival rates of vitrification and quick-freezing (84.5 and 74.3%, respectively) were similar to the slow-freezing. In vivo, the implantation rate and number of fetuses from embryos frozen through slow-freezing at 1.2ºC/minute, vitrification and quick-freezing were not significantly different.

Os efeitos das temperaturas de imersão em nitrogênio líquido e dos métodos de remoção dos crioprotetores foram avaliados em mórulas de camundongos congeladas em etilenoglicol (E), propilenoglicol (P) e glicerol (G). Os embriões foram equilibrados em E (1,5M), P (1,5M) ou G (1,4M) por 10 minutos e envasados em palhetas de 0,25 ml com crioprotetor nas três colunas (E1, P1 G1) ou PBS nas colunas das extremidades (E2, P2). As palhetas foram resfriadas a 0,5ºC/minuto até -25 ou -30ºC e imersas em nitrogênio líquido. A descongelação dos embriões foi feita em água a 22ºC por 20 segundos. O crioprotetor dos embriões congelados em glicerol (Gl) foi removido em 3 etapas, dos congelados em etilenoglicol e propilenoglicol com crioprotetor nas 3 colunas (El e Pl) removido diretamente em PBS e dos congelados em etilenoglicol e propilenoglicol com PBS nas colunas das extremidades (E2, P2), após mistura das três colunas dentro da palheta, em PBS. Não houve influência da temperatura de imersão sobre o desenvolvimento embrionário in vitro, observando maior taxa (p < 0,0001) para El (69,2%) que para E2 (60,3%), Gl (51,9%) e a combinação P1 e P2 (46,9%). Para o desenvolvimento in vivo, a taxa de fetos foi menor (p < 0,07) para o grupo E1-25 do que para o Controle; Gl-30ºC; El-30ºC e E2-25ºC. Pode-se concluir que in vitro o melhor crioprotetor foi o etilenoglicol com remoção direta em PBS e que in vivo o etilenoglicol e o glicerol foram semelhantes a -30ºC.

The effects of plunging temperature in liquid nitrogen and cryoprotectant dilution methods were evaluated for compacted mouse morulae frozen in 1.5 M ethylene-glycol (E), 1.5M propylene-glycol (P) or 1.4M glycerol (G). Morulae were equilibrated for 10 minutes in cryoprotectant solution and loaded into 0.25 ml straws with cryoprotectant solution in 3 columns (groups E1, P1, G1) or cryoprotectant in the center and PBS in the lateral columns (E2, P2). Straws were cooled at 0.5ºC/min to -25 or -30ºC and plunged into liquid nitrogen. Straws were thawed in water at 22ºC for 20 seconds. Cryoprotectant was diluted in 3 steps for group G1 and in one step for groups E1 and P1 (direct transfer to PBS + 10% FCS) and E2 and P2 (shaken to mix the 3 columns before transferring to PBS+ 10% FCS). Plunging temperature had no significant effect on the proportion of morulae developed to blastocysts in vitro; this proportion was higher (p < 0.0001) in E1 (69.2%) than in E2 (60.3%), G1 (51.9%) and combined for P1 and P2 (46.9%). In second experiment, the proportion of transferred morulae that developed to viable fetuses was lower (p < 0.07) for E1-25 than for E1-30, G1-30, E2-25 or unfrozen (control) embryos (8.7, 20.0, 20.0, 17.4 and 19.8%, respectively). In conclusion, the ethylene glycol diluted directly in PBS (E1) exhibit the highest rate of in vitro embryos development, but based on in vivo embryos development was more efficacious in plunging temperature at -30ºC (E1-30).