Genome-wide association study of Nelore and Angus heifers with low and high ovarian follicle counts.

The number of antral follicles is considered an important fertility trait because animals with a high follicle count (HFC) produce more oocytes and embryos per cycle. Identification of these animals by genetic markers such as single nucleotide polymorphisms (SNPs) can accelerate selection of future generations. The aim of this study was to perform a genome wide association study (GWAS) on Nelore and Angus heifers with HFC and low (LFC) antral follicle counts. The groups HFC and LFC for genotyping were formed based on the average of total follicles (≥ 3 mm) counted in each breed consistently ± standard deviation. A total of 72 Nelore heifers (32 HFC and 40 LFC) and 48 Angus heifers (21 HFC and 27 LFC) were selected and the DNA was extracted from blood and hair bulb. Genotyping was done using the Illumina Bovine HD 770K BeadChip. The GWAS analysis showed 181 and 201 SNPs with genotype/phenotype association (P ≤ 0.01) in Nelore and Angus heifers, respectively. Functional enrichment analysis was performed on candidate genes that were associated with SNPs. A total of 97 genes were associated to the 181 SNPs in the Nelore heifers and the functional analysis identified genes (ROBO1 and SLIT3) in the ROBO-SLIT pathway that can be involved in the control of germ cell migration in the ovary as it is involved in lutheal cell migration and fetal ovary development. In the Angus heifers, 57 genes were associated with the 201 SNPs, highlighting Fribilin 1 (FBN1) gene, involved in regulation of growth factors directly involved in follicle activation and development. In summary, GWAS for Nelore and Angus heifers showed SNPs associated with higher follicle count phenotype. Furthermore, these findings offer valuable insights for the further investigation of potential mechanism involved in follicle formation and development, important for breeding programs for both breeds.

Effects of heat stress on sperm quality of French Bulldogs.

This study aimed to evaluate the effects of heat stress (HS) and its duration on semen quality, serum testosterone, pulsatility and resistibility index of the testicular artery of French Bulldogs. Eight male French Bulldogs, 3-7 years old, 12.63 ± 1.8 Kg were adapted and trained for two months. Room temperature was 21 °C. Semen was collected by digital stimulation. The median of four andrological evaluations was T0. Heat was applied to the scrotum using an electrical heat pad at 40 °C for 11 min. Rectal temperature (RT) and scrotum temperature were evaluated using a mercury thermometer and an infrared thermography camera before and after HS. Semen was evaluated immediately (T1) and after seven (T7), 14 (T14), 21 (T21), 28 (T28) and 60 (T60) days after HS. Semen parameters included macroscopic (volume, color and viscosity) and microscopic (sperm motility and vigor, percentage of morphologically normal or defected spermatozoa, sperm concentration and total number of sperm cells) aspects. A pulsed colored doppler ultrasound was performed on the testicular artery at the spermatic cord and epididymis region before and immediately after HS. Serum testosterone was analyzed before, 48 and 96 h after HS. Data was analyzed by ANOVA using SAS. There was a 1.23 °C increase on RT and a 4.98 °C increase on thermograph after HS. Sperm motility decreased at T1 (P < 0.05) and tended to stay lower at T7 (P = 0.056). It improved at T14, but reduced again at T21 (P < 0.05). At T28 and T60 motility was normal. Vigor was lower at T1 (P < 0.05), normal at T7 and T14, but decreased at T21 (P = 0.054), at T28 and T60 it was not different than T0. Sperm concentration was lower at T1 (P < 0.05) and not different from T0 at other timepoints. Volume color and viscosity were not different. Total sperm per ejaculate was reduced at T1 and T7 (P < 0.05) and tended to be lower at T14 (P = 0.057). T21, T28 and T60 were not different than T0. There was a decrease in normal sperm cells and an increase in defected sperm at T7. There was no difference within T14, T21, T28 and T60. The raise in pathologies at T7 was from an increase in minor defects (P < 0.05). There was no difference in serum concentration of testosterone nor pulsatility and resistivity index before and after HS. In conclusion, induction of HS directly to the testis reduces sperm quality in French Bulldog. This impairment is immediately and transitory.

Influência da progesterona injetável na taxa de prenhez e perda gestacional de fêmeas nelore de distintas categorias reprodutivas submetidas a inseminação artificial em tempo fixo / Influence of injectable progesterone on the pregnancy rate and gestational loss of Nellore females of different reproductive categories undergoing fixed-time artificial insemination

Progesterone (P4) is essential for embryonic development and maintenance of pregnancy when deficiency causes early embryonic loss. In this study, we investigated the ability of hormonal supplementation to improve the fertility of Nellore females subjected to fixed-time artificial insemination (IATF) protocols. Here, we evaluated the effect of long-acting injectable progesterone (iP4) supplementation in the D4 after IATF on pregnancy rate and pregnancy loss in Nellore females (Bos taurus indicus) from different reproductive categories in Western Amazonia. Eight hundred thirteen Nellore females from 5 farms were selected and distributed into 2 groups: control [GC; administration of 0.5 mL of 0.9% saline solution, intramuscularly - IM] (n = 407) and a group that received injectable progesterone (iP4) that was long-acting [GiP4; administration of 0.5 mL of iP4, 300 mg, via IM four days after IATF] (n = 406). Each group contained 3 subgroups: heifers, primiparous cows, and multiparous cows. Of the 407 animals in the CG, 103 were heifers, 107 primiparous, and 197 multiparous. Of the 406 animals in the GiP4 group, there were 101 heifers, 107 primiparous, and 198 multiparous. On a random day of the estrous cycle (D0), an intravaginal device containing 1 g of P4 and 2 mg of estradiol benzoate (BE) was inserted by intramuscular injection. On D8, the P4 device was removed and 150 µg of D-cloprostenol (PGF2α), 300 IU eCG, and 1 mg BE were administered IM. Cows were inseminated at D10, 48-52 h after procedure on D8. Pregnancy diagnosis was made between 35 and 40 days after insemination through ultrasound examination. Between 80 and 90 days after insemination, a new ultrasound examination was performed to assess early pregnancy loss. The data were processed using the SAS 9.2. The conception rate, pregnancy loss, and final conception rate were analyzed using PROC GLIMMIX according to groups (CG and GiP4), categories (heifers, primiparous and multiparous), and their interactions as variables. The differences in the means of least squares were adjusted using the Tukey­Kramer method. Statistical significance was defined as P < 0.05. The general conception rate was 46% (375/816). Regardless of the animal class, GiP4 animals (51.97%) had higher conception rates (P < 0.05) than CG (40.29%). In the subgroups (heifers, primiparous and multiparous cows), there was a difference (P < 0.05) between animals treated with iP4 (52.48%, 57.94%, and 48.48%, respectively) and those who were not (39.81%, 41.12%, and 40.10%, respectively). Gestational losses, regardless of the animal class, were higher in females in the CG (7.93%) [P < 0.05] compared to those in the GiP4 group (2.84%). Regardless of treatment with iP4, the percentage of gestational loss in heifers was significantly higher (10.64%) than that in primiparous and multiparous cows (3.77% and 2.86%, respectively). The final conception rates were higher in animals that received long-acting iP4, which increased the final pregnancy in all parity categories. In the present study, the use of iP4 increased the pregnancy rate in Nellore females, regardless of the category. Although there has been no consensus on the use of iP4, there is an agreement that increases in the pregnancy rate are related to the moment of exogenous P4 application. In addition to influencing the pregnancy rate, reduction in pregnancy losses is also attributed to iP4 treatment, a fact demonstrated in the present study, where animals treated with iP4 had a lower pregnancy loss rate than normally occurs in beef cattle. Supplementation with long-acting iP4 increased the pregnancy rate at D35-40, reduced pregnancy losses, and increased the conception rate on D80-90 days in Nellore females reared in the Western Amazon, regardless of the animal category.(AU)

Sodium caseinate improves longevity and fertility of frozen bull semen.

This study aimed to evaluate the effect of sodium caseinate added into freezing extender on the sperm parameters of cryopreserved bull semen and in vitro and in vivo fertility. One ejaculate of 30 bulls was used and processed using Botu-Bov (Botupharma, Botucatu, Brazil) with the addition of 20% egg yolk (EY) or 15% egg yolk with 2% sodium caseinate (EY + SC), subsequently submitted to freezing. Semen from both groups were evaluated immediately after thawing (T0) and after thermic stress at 37 °C for 90 min (T90), for sperm kinetics, by CASA method, and plasma membrane integrity (PMI), superoxide (O2-) concentration and high mitochondrial potential (HMP) by flow cytometry. In vitro fertilization (IVF) was performed to assess embryo cleavage rate on day 3, and blastocyst rate on day 8. The in vivo fertility test was performed using fixed-time artificial insemination (FTAI). In sperm evaluation, trajectory velocity, linear velocity, curvilinear velocity, and lateral head movement were higher (P < 0.05) in EY + SC at T0. At T90, while rectilinearity and linearity did not differ between EY and EY + SC (P > 0.05), the other parameters evaluated were higher in EY + SC. Similarly, the integrity of the plasma and acrosomal membranes (iPAM) was higher (P < 0.05) at T90 in EY + SC, but did not differ (P > 0.05) between the groups at T0. For O2- and HMP, the values were lower (P < 0.05) in EY + SC group in both moments; furthermore, EY + SC showed higher cleavage and blastocyst rates in IVF. Likewise, pregnancy rates by FTAI were higher (P < 0.05) in the EY + SC group. In conclusion, the addition of sodium caseinate into freezing extender improves sperm parameters of frozen-thawed bull semen and fertility rates on during in vitro and in vivo tests.

Nematicidal activity of silver nanoparticles from the fungus Duddingtonia flagrans.

BACKGROUND: Helminth parasites cause morbidity and mortality in both humans and animals. Most anthelmintic drugs used in the treatment of parasitic nematode infections act on target proteins or regulate the electrical activity of neurons and muscles. In this way, it can lead to paralysis, starvation, immune attack, and expulsion of the worm. However, current anthelmintics have some limitations that include a limited spectrum of activity across species and the threat of drug resistance, which highlights the need for new drugs for human and veterinary medicine. PURPOSE: Present study has been conducted to determine the anthelmintic activity of silver nanoparticles (AgNPs) synthesized from the extract of nematophagous fungus, Duddingtonia flagrans, on the infecting larvae of Ancylostoma caninum (L3). METHODS: The nanoparticles were characterized by visual, ultraviolet, Fourier-transform infrared spectroscopy, transmission electron microscopy (TEM) analysis, and X-ray diffraction. The in vitro study was based on experiments to inhibit the motility of infective larvae (L3), and the ultrastructural analysis of the nematode was performed by images obtained by TEM. RESULTS: The XRD studies revealed the crystalline nature of the nanoparticles, and FTIR results implied that AgNPs were successfully synthesized and capped with compounds present in the extract. The results showed that the green synthesis of AgNPs exhibited nematicidal activity, being the only ones capable of penetrating the cuticle of the larvae, causing changes in the tegmentum, and consequently, the death of the nematode. CONCLUSION: The extract of the fungus D. flagrans is able to synthesize AgNP and these have a nematicidal action.

Evolução clínica e nutricional de uma paciente com a síndrome de hallervorden spatz: relato de caso clínico / Clinical and nutritional evolution of a patient with hallervorden spatz syndrome: clinical case report

Relatou-se o caso de um paciente com a Síndrome de Hallervorden Spatz, acolhida no programa domiciliar do Hospital Regional da Asa Norte (HRAN), Brasília, DF e discutir a terapia nutricional instituída para melhoria de qualidade de vida da mesma. Trata-se de um estudo de caso clínico compreendido entre o período de 01/09/2017 a 30/10/2017 onde se averiguou toda a evolução clínica e nutricional do paciente durante o período de internação. Após a introdução adequada de uma conduta dietoterápica para a situação da paciente, ocorreram mudanças em seu diagnóstico nutricional, levando-a a sair do nível de desnutrição com consequente ganho ponderal, além de aliviar a constipação intestinal crônica e promover a cicatrização da lesão por pressão. A partir de uma elaboração dietoterápica, pode-se notar que a terapia nutricional instituída demonstrou benefícios para a qualidade de vida da paciente com a Síndrome de Hallervorden Spatz

Effects of three methadone doses combined with acepromazine on sedation and some cardiopulmonary variables in dogs.

OBJECTIVE: To evaluate the sedative and cardiopulmonary effects of three methadone doses, combined with acepromazine, in dogs. STUDY DESIGN: Prospective, randomized, complete block study. ANIMALS: Six healthy, adult, cross-bred dogs weighing 17.2±4.4 kg (mean±standard deviation). METHODS: Each dog was administered four treatments: acepromazine (0.05 mg kg-1) alone or acepromazine (same dose) in combination with methadone (0.25, 0.50 or 0.75 mg kg-1). All drugs were administered intramuscularly. Sedation was scored by a numeric descriptive scale (NDS, range 0-3) and a simple numerical scale (SNS, range 0-10). Heart rate, invasive blood pressure, arterial blood gases and rectal temperature were measured at 15 to 30 minute intervals for 120 minutes. RESULTS: According to NDS scores, mild to moderate sedation (NDS=1-2) was observed in most dogs in the acepromazine treatment, with only one out of six dogs scored as exhibiting intense sedation (NDS=3). All treatments with methadone resulted in significantly higher SNS scores compared with acepromazine alone. In these treatments, most dogs exhibited intense sedation (NDS=3). Increasing the dose of methadone from 0.25 to 0.50 or 0.75 mg kg-1 prolonged sedation in a dose-related manner, but did not influence the degree of sedation. The main adverse effects following administration of acepromazine-methadone treatments were decreased blood pressure, mild respiratory acidosis and decreased rectal temperature. These effects were well tolerated and resolved without treatment. CONCLUSIONS AND CLINICAL RELEVANCE: In this study in six dogs, acepromazine-methadone administration resulted in intense sedation in most dogs. The results are interpreted to indicate that a low dose of methadone (0.25 mg kg-1) administered in combination with acepromazine (0.05 mg kg-1) will induce short-term sedation in dogs, whereas higher doses of methadone should be administered when prolonged sedation is desired.

Consequences of eight consecutive applications of equine chorionic gonadotropin on ovarian follicle development and ovulation in bos indicus cows / Consequências do uso consecutivo de oito doses de gonadotrofina coriônica equina no desenvolvimento folicular e ovulação de vacas bos indicus

ABSTRACT: The equine chorionic gonadotropin (eCG) is a glycoprotein produced in mare endometrial calices. In bovine, it is used in estrus synchronization protocols. However, studies have shown that it is potentially immunogenic and its effect can decrease after repetitive use. This study aimed to evaluate antral follicle dynamics, corpus luteum (CL) and ovulation rate in bos indicus cows submitted to an estrus synchronization protocol in association with eCG for eight times consecutively. Ten cyclical, multiparous, and pasture raised beef cows were divided into two groups: control group (n=5) and eCG group (n=5). In 30 day interval, all animals were synchronized with the same estrogen/progesterone based protocol, totalizing 8 re-synchronizations. Cows in the treatment group received 300IU eCG 48 hours prior to the presumable ovulation. Ultrassound examinations were performed on Day 4 of the protocol (approximately 1.5 days after follicle recruitment) to count antral follicles, on Day 10, to count antral follicles and to measure size of the largest follicle and on Day 18 to measure the diameter of the CL. No difference (P>0.05) between follicular growth and size of the pre-ovulatory follicle was reported between groups. Cows treated with eCG had a larger (P<0.05) CL and increased (P<0.05) ovulation rate (18mm and 92%, respectively) when compared with control group (14.1mm and 80%, respectively). Furthermore, consecutive treatments did not affect CL nor ovulation rates. In conclusion, eCG treatment increased CL size and ovulation rate even after 8 consecutive treatments.

RESUMO: A gonadotrofina coriónica equina (ECG) é uma glicoproteína produzida nos cálices endometriais da égua. Em bovinos, é utilizada em protocolos de sincronização do estro. Estudos têm mostrado que ela é potencialmente imunogênica e seu efeito pode diminuir depois de utilização repetida. O objetivo do presente trabalho foi avaliar a dinâmica folicular, o diâmetro do corpo lúteo (CL) e taxa de ovulação em vacas bos indicus submetidas a um protocolo de sincronização estral com eCG por oito vezes. Dez vacas de corte, cíclicas, multíparas e solteiras foram divididas em dois grupos: grupo controle (n=5) e grupo eCG (n=5). Em um intervalo de 30 dias, os animais foram sincronizados com um protocolo de estrógeno/progesterona, totalizando 8 re-sincronizações. Os animais do grupo tratamento receberam 300UI eCG 48 horas antes da provável ovulação. Exames ultrasonográficos foram realizados no dia 4 do protocolo (aproximadamente 1,5 dias após o recrutamento folícular), para contar folículos antrais, no dia 10, contar folículos antrais e medir o tamanho do maior folículo e, no dia 18, para medir o diâmetro do CL. Não foi encontrada diferença (P>0,05) entre folículos no dia 4 ou 10. Vacas tratadas com eCG tinham um maior (P<0.05) CL e elevada (P<0.05) taxa de ovulação (18mm e 92%, respectivamente) quando comparadas ao grupo controle (14,1mm e 80%, respectivamente). Além disso, tratamentos consecutivos não afetaram o CL nem as taxas de ovulação. Em conclusão, o tratamento com eCG aumentou o tamanho do CL e taxa de ovulação, mesmo depois de 8 tratamentos consecutivos.

Expression of androgen-producing enzyme genes and testosterone concentration in Angus and Nellore heifers with high and low ovarian follicle count.

Follicle population is important when animals are used in assisted reproductive programs. Bos indicus animals have more follicles per follicular wave than Bos taurus animals. On the other hand, B taurus animals present better fertility when compared with B indicus animals. Androgens are positively related with the number of antral follicles; moreover, they increase growth factor expression in granulose cells and oocytes. Experimentation was designed to compare testosterone concentration in plasma, and follicular fluid and androgen enzymes mRNA expression (CYP11A1, CYP17A1, 3BHSD, and 17BHSD) in follicles from Angus and Nellore heifers. Heifers were assigned into two groups according to the number of follicles: low and high follicle count groups. Increased testosterone concentration was measured in both plasma and follicular fluid of Angus heifers. However, there was no difference within groups. Expression of CYP11A1 gene was higher in follicles from Angus heifers; however, there was no difference within groups. Expression of CYP17A1, 3BHSD, and 17BHSD genes was higher in follicles from Nellore heifers, and expression of CYP17A1 and 3BHSD genes was also higher in HFC groups from both breeds. It was found that Nellore heifers have more antral follicles than Angus heifers. Testosterone concentration was higher in Angus heifers; this increase could be associated with the increased mRNA expression of CYP11A1. Increased expression of androgen-producing enzyme genes (CYP17A1, 3BHSD, and 17BHSD) was detected in Nellore heifers. It can be suggested that testosterone is acting through different mechanisms to increase follicle development in Nellore and improve fertility in Angus heifers.

Exposure to colony stimulating factor 2 during preimplantation development increases postnatal growth in cattle.

The microenvironment of a preimplantation embryo can influence changes in development that affect postnatal phenotypes. One of the potential mediators of this effect in many species is colony-stimulating factor (CSF2), which can increase an embryo's ability to establish pregnancy after its transfer into recipients. Exposure of embryos to CSF2 during early development can also affect the pattern of development later in pregnancy in a sex-dependent manner. We therefore hypothesized that treatment of in vitro-produced embryos with CSF2 in culture would alter birth weight and postnatal growth of the resultant calf. Body weight and withers height were measured for Holstein heifer calves produced in vitro with or without 10 ng/ml CSF2 and for calves produced by artificial insemination. There were no differences in birth weight between groups; thereafter, however, calves from the CSF2-treated group experienced greater increases in body weight through 13 months of age, with only small differences in withers height. These results support the model that an embryo's postnatal characteristics can be programmed during the preimplantation period, and that CSF2 is one of the embryokines through which programming is directed. Mol. Reprod. Dev. 82: 892-897, 2015. © 2015 Wiley Periodicals, Inc.

Sexual dimorphism in developmental programming of the bovine preimplantation embryo caused by colony-stimulating factor 2.

Physiology of the adult can be modified by alterations in prenatal development driven by the maternal environment. Developmental programming, which can be established before the embryo implants in the uterus, can affect females differently than males. The mechanism by which sex-specific developmental programming is established is not known. Here we present evidence that maternal regulatory signals change female embryos differently than male embryos. In particular, actions of the maternally derived cytokine CSF2 from Day 5 to Day 7 of development affected characteristics of the embryo at Day 15 differently for females than males. CSF2 decreased length and IFNT secretion of female embryos but increased length and IFNT secretion of male embryos. Analysis of a limited number of samples indicated that changes in the transcriptome and methylome caused by CSF2 also differed between female and males. Thus, sex-specific programming by the maternal environment could occur when changes in secretion of maternally derived regulatory molecules alter development of female embryos differently than male embryos.

Canonical WNT signaling regulates development of bovine embryos to the blastocyst stage.

Objectives were to evaluate the role of canonical WNT signaling in development of the preimplantation embryo. Signaling was activated with 2-Amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine (AMBMP) and inhibited with Dickkopf-related protein 1 (DKK1). Treatment of bovine embryos with AMBMP at day 5 after insemination decreased development to the blastocyst stage at day 7 and reduced numbers of trophectoderm and inner cell mass cells. At high concentrations, AMBMP caused disorganization of the inner cell mass. DKK1 blocked actions of AMBMP but did not affect development in the absence of AMBMP. Examination of gene expression in day 6 morulae by microarray revealed expression of 16 WNT genes and other genes involved in WNT signaling; differences in relative expression were confirmed by PCR for 7 genes. In conclusion, the preimplantation embryo possesses a functional WNT signaling system and activation of the canonical pathway can inhibit embryonic development.

Evaluation of the hypothalamus-pituitary axis response to exogenous GnRH, estradiol benzoate, and LH during the postpartum period in Nellore cows.

The objective was to evaluate when the LH reserve was re-established in postpartum Nellore (Bos indicus) cows by evaluating the response of the hypothalamic-pituitary axis responsiveness to exogenous GnRH or estradiol benzoate (EB). Additionally, we tested the influence of dietary supplementation (SUPL) and calf removal (CR) on the duration of postpartum anestrus. Ninety multiparous lactating Nellore cows were randomly assigned to eight groups. The EB and GnRH groups received 1.0 mg EB (N = 7), and 50 µg lecireline (N = 16), respectively. Additional cows were given the same hormones, and subjected to either nutritional supplementation (EB-SUPL, N = 9; GnRH-SUPL, N = 16), or calf removal at 72 hours after calving (EB-CR, N = 4; GnRH-CR, N = 13). The remaining two groups were the LH (12.5 mg, N = 14) and control groups (saline, N = 11). Hormones were administered weekly from 7 (±5) days postpartum to first ovulation (detection of a CL during a weekly ultrasonographic examination). Blood samples were collected just before and 2 hours (GnRH, LH, and control groups) or 18 hours (EB groups) after hormone or saline (control) administration. Ovulation occurred as early as 15 days postpartum in the GnRH group. The mean ± SEM intervals (days) from calving to first ovulation were EB, 87.7 ± 4.2; EB-CR, 20.3 ± 1.2; EB-SUPL, 60.3 ± 3.2; GnRH, 40.4 ± 2.1; GnRH-CR, 21.0 ± 1.1; GnRH-SUPL, 26.4 ± 1.1; LH, 35.6 ± 1.1; and control, 60.9 ± 2.1. We concluded that there was sufficient LH in the pituitary gland (of Nellore cows) from the second week postpartum to induce ovulation in response to exogenous GnRH. Additionally, calf removal and nutritional supplementation reduced, by 2 to 4 weeks, the interval from calving to an LH increase and ovulation induced by GnRH or EB.

Consequences of conceptus exposure to colony-stimulating factor 2 on survival, elongation, interferon-τ secretion, and gene expression.

Exposure of bovine conceptuses to colony-stimulating factor 2 (CSF2) from days 5 to 7 of development can increase the percentage of transferred conceptuses that develop to term. The purpose of this experiment was to understand the mechanism by which CSF2 increases embryonic and fetal survival. Conceptuses were produced in vitro in the presence or absence of 10  ng/ml CSF2 from days 5 to 7 after insemination, transferred into cows, and flushed from the uterus at day 15 of pregnancy. There was a tendency (P=0.07) for the proportion of cows with a recovered conceptus to be greater for those receiving a CSF2-treated conceptus (35% for control versus 66% for CSF2). Antiviral activity in uterine flushings, a measure of the amount of interferon-τ (IFNT2) secreted by the conceptus, tended to be greater for cows receiving CSF2-treated conceptuses than for cows receiving control conceptuses. This difference approached significance when only cows with detectable antiviral activity were considered (P=0.07). In addition, CSF2 increased mRNA for IFNT2 (P=0.08) and keratin 18 (P<0.05) in extraembryonic membranes. Among a subset of filamentous conceptuses that were analyzed by microarray hybridization, there was no effect of CSF2 on gene expression in the embryonic disc or extraembryonic membranes. Results suggest that the increase in calving rate caused by CSF2 treatment involves, in part, more extensive development of extraembryonic membranes and capacity of the conceptus to secrete IFNT2 at day 15 of pregnancy.

Colony-stimulating factor 2 inhibits induction of apoptosis in the bovine preimplantation embryo.

PROBLEM: Addition of colony-stimulating factor 2 (CSF2) to culture medium increases post-transfer survival of bovine embryos. Here we provide evidence that one mechanism by which CSF2 affects the embryo is through inhibition of apoptosis. METHOD OF STUDY: In the first experiment, genes and pathways whose expression were regulated by CSF2 were identified by microarray analysis. Embryos were treated with 10 ng/ml CSF2 or vehicle at Day 5 after insemination; morulae were selected for microarray analysis at Day 6. In a second experiment, antiapoptotic effects of CSF2 were determined. Embryos were treated with CSF2 or vehicle at Day 5. On Day 6 (24 h after treatment), morulae were cultured for 15 h at either 42°C (a temperature that induces apoptosis) or 38.5°C (cow body temperature). RESULTS: In the first experiment, a total of 214 genes were differentially regulated and 160 of these could be annotated (67 upregulated genes and 93 downregulated genes). Differentially expressed genes could be placed in 13 biological process ontologies in four functional groups (development and differentiation process, cell communication, apoptosis and cell adhesion). Antiapoptotic effects of CSF2 were confirmed in the second experiment because the magnitude of the increase in TUNEL positive cells caused by heat shock was reduced by CSF2. CONCLUSION: CSF2 blocks apoptosis in bovine embryos through actions associated with regulation of genes controlling apoptosis.

Effects of gamete source and culture conditions on the competence of in vitro-produced embryos for post-transfer survival in cattle.

One limitation to the use of in vitro-produced embryos in cattle production systems is the fact that pregnancy rates after transfer to recipients are typically lower than when embryos produced in vivo are transferred. Conceptually, the oocyte and spermatozoon from which the embryo is derived could affect competence for post-transfer survival. There are sire differences in embryonic survival after transfer, but there is little evidence that an embryo's ability to establish pregnancy is determined by sex sorting of spermatozoa by flow cytometry. The role of the source of the oocyte as a determinant of embryonic survival after transfer has not been examined carefully. Conditions for embryo culture after fertilisation can have an impact on the ability of the embryo to establish pregnancy following transfer. Among the specific molecules produced in the reproductive tract of the cow that have been shown to improve competence of in vitro-produced embryos for post-transfer survival are colony-stimulating factor 2, insulin-like growth factor-1 (for recipients exposed to heat stress) and hyaluronan (for less-advanced embryos). There is also a report that embryo competence for post-transfer survival can be improved by inclusion of a carbon-activated air filtration system in the incubator used to culture embryos. Progress in developing culture systems to improve embryonic competence for survival after transfer would be hastened by the development of in vitro assays that accurately predict the potential of an embryo to establish pregnancy after transfer. A group of 52 genes has been identified that are differentially expressed in embryos that developed to term v. embryos that did not establish pregnancy. Perhaps a gene microarray consisting of these genes, alone or in combination with other genes, could be used to screen embryos for competence to establish pregnancy.

Colony-stimulating factor 2 (CSF-2) improves development and posttransfer survival of bovine embryos produced in vitro.

In this study, we tested the role of colony-stimulating factor 2 (CSF2) as one of the regulatory molecules that mediate maternal effects on embryonic development during the preimplantation period. Our objective was to verify effects of CSF2 on blastocyst yield, determine posttransfer survival, and evaluate properties of the blastocyst formed after CSF2 treatment. In vitro, CSF2 increased the percentage of oocytes that became morulae and blastocysts. Blastocysts that were treated with CSF2 tended to have a greater number of inner cell mass cells and had a higher ratio of inner cell mass to trophectoderm cells. There was no effect of CSF2 on the incidence of apoptosis. Treatment with CSF2 from d 5 to 7 after insemination increased embryonic survival as indicated by improved pregnancy rate at d 30-35 of gestation. Moreover, treatment with CSF2 from either d 1-7 or 5-7 after insemination reduced pregnancy loss after d 30-35. Results indicate that treatment with CSF2 can affect embryonic development and enhance embryo competence for posttransfer survival. The fact that treatment with CSF2 during such a narrow window of development altered embryonic function much later in pregnancy suggests that CSF2 may exert epigenetic effects on the developing embryo that result in persistent changes in function during the embryonic and fetal periods of development.

Heat shock and tumor necrosis factor-alpha induce apoptosis in bovine preimplantation embryos through a caspase-9-dependent mechanism.

Heat shock and tumor necrosis factor-alpha (TNF-alpha) induce apoptosis through different mechanisms, with heat shock acting to cause mitochondrial depolarization and caspase-9 activation, while TNF-alpha acts through a receptor-mediated process to activate caspase-8. In some cells, however, TNF-alpha can also cause mitochondrial depolarization and caspase-9 activation. In the present study, we tested the hypothesis that heat shock at 41 degrees C and TNF-alpha induce apoptosis in bovine preimplantation embryos through a caspase-9-dependent mechanism. Treatment of embryos with either heat shock (41 degrees C) or TNF-alpha increased the proportion of blastomeres that were TUNEL positive and the proportion of embryos exhibiting elevated caspase-9 activity. Furthermore, the caspase-9 inhibitor, z-LEHD-fmk, blocked the increase in TUNEL-positive nuclei caused by both heat shock and TNF-alpha. For embryos at day 6 after insemination, for example, the percent of blastomeres positive for TUNEL was 3.6% for control embryos, 11.1% for embryos cultured at 41 degrees C, and 15.1% for embryos cultured with 10 ng/ml TNF-alpha. In the presence of z-LEHD-fmk, the percent of cells positive for TUNEL was 3.7% for control embryos, 6.1% for embryos cultured at 41 degrees C, and 8% for embryos cultured with 10 ng/ml TNF-alpha. Although TNF-alpha did not cause a measurable increase in caspase-8 activity, there was a tendency (P = 0.07) for treatment of embryos with z-IETD-fmk, an inhibitor of caspase-8, to partly reduce the magnitude of the increase in TUNEL-positive cells caused by TNF-alpha. The percent of cells that were TUNEL positive was increased by TNF-alpha from 9.7 to 19.7% in the absence of inhibitor and from 13.0 to 15.6% in the presence of z-IETD-fmk. Results indicate that induction of apoptosis by both heat shock and TNF-alpha involve activation of caspase-9-dependent pathways. It is likely that TNF-alpha also activates apoptotic pathways involving caspase-8 but that the degree of activation is small and caspase-9-dependent pathways are required for full activation of apoptosis.