Thermal-treatment protocol to induce thermotolerance in bovine embryos.

Artificial reproduction in dairy cattle is challenged by summer temperatures in tropical environments. We describe a treatment based on mild temperature increases to induce thermotolerance and improve the embryo's performance under heat stress conditions. A protocol was established to induce upregulation of heat shock protein A (HSPA, formerly known as HSP70) but not impair embryonic development. Thermal treatment (TT) had no effect on morula/blastocyst rate or blastocyst quality (cell number and apoptosis). Heat shock given one day after TT revealed higher (P=0.00) survival rates in TT blastocysts compared with Control. Treated embryos were transferred to recipients and no detrimental effects were observed regarding pregnancy rates, length, fetal growth or calf weight. Our results demonstrated that the established TT protocol could induce a thermal response by the embryo and is safe for further development.

In-straw warming protocol improves survival of vitrified embryos and allows direct transfer in cattle.

Vitrification is a superior method for cryopreservation of IVF embryos, but due to complicated warming protocols, it is not commonly used for commercial bovine embryos routine. To overcome the need of laboratory embryo preparation during warming, we developed an in-straw warming protocol compatible with most vitrification devices for embryo transfer without sucrose gradient steps and embryo evaluation. Surprisingly, one of the tested protocols improved embryo survival (95.0%* vs 83.1% expansion rate and 74.2%* vs 51.5% hatching rate) compared to conventional in-plate warming. Embryo quality was also increased, taken by the higher total cell numbers (160.7 ± 8.6* vs 99.0 ± 7.9) and lower apoptosis index (4.9 ± 0.6* vs 11.5 ± 2.4) 48 h after warming. Pregnancy rates were similar between vitrified-warmed embryos and fresh embryos (40% vs 43%). Based on our results, we suggest in-straw warming should always be used for vitrified embryos due to beneficial effects. Direct transfer can be safely performed using this protocol.

Oocyte origin affects the in vitro embryo production and development of Holstein (Bos taurus taurus) - Gyr (Bos taurus indicus) reciprocal cross embryos.

A reciprocal crossbred embryo production approach was used to assess effects of maternal breed on embryo development in tropical conditions (average temperature 22.0 °C and 77.9% relative humidity). Oocytes were recovered by ovum pick-up (OPU) from Gyr and Holstein donors (n = 90 Holstein and 83 Gyr OPUs). Female F1 embryos were produced by fertilization with sperm bearing X-chromosomes from Holstein semen (n = 615 Gyr oocytes) or Gyr semen (n = 255 Holstein oocytes). Blastocysts were transferred to recipients 168 h post-insemination (h.p.i.) (n = 70-144) and there were assessments of pregnancies until birth. Oocyte number per OPU (Gyr 10.0 ±â€¯0.7 compared with Holstein 6.3 ±â€¯0.4) and percentage viable oocytes (Gyr 78.8 ±â€¯1.9% compared with Holstein 71.2 ±â€¯2.2%) were less for Holstein donor animals. There was a 2.8 fold fewer total number of F1 blastocysts when Holstein donors were used (Gyr: 260, Holstein: 91). Pregnancy assessment during the different stages of gestation indicated the percentage pregnancy was less when embryos were produced from Holstein oocytes (Gyr and Holstein respectively: early pregnancy, 47.9% compared with 38.6%; mid-pregnancy, 44.4% compared with 31.4%; late pregnancy, 41.0% compared with 22.9%). Pregnancy length was also affected by maternal breed (Gyr: 280.8 ±â€¯0.6, Holstein: 286.3 ±â€¯0.7). It is concluded that in a tropical environment the maternal breed affects crossbred embryo development with pregnancy rates during the latter stages of gestation being greater when Gyr oocytes are used for production of embryos.

Genome-wide association studies for heat stress response in Bos taurus × Bos indicus crossbred cattle.

Heat stress is an important issue in the global dairy industry. In tropical areas, an alternative to overcome heat stress is the use of crossbred animals or synthetic breeds, such as the Girolando. In this study, we performed a genome-wide association study (GWAS) and post-GWAS analyses for heat stress in an experimental Gir × Holstein F2 population. Rectal temperature (RT) was measured in heat-stressed F2 animals, and the variation between 2 consecutive RT measurements (ΔRT) was used as the dependent variable. Illumina BovineSNP50v1 BeadChip (Illumina Inc., San Diego, CA) and single-SNP approach were used for GWAS. Post-GWAS analyses were performed by gene ontology terms enrichment and gene-transcription factor (TF) networks, generated from enriched TF. The breed origin of marker alleles in the F2 population was assigned using the breed of origin of alleles (BOA) approach. Heritability and repeatability estimates (± standard error) for ΔRT were 0.13 ± 0.08 and 0.29 ± 0.06, respectively. Association analysis revealed 6 SNP significantly associated with ΔRT. Genes involved with biological processes in response to heat stress effects (LIF, OSM, TXNRD2, and DGCR8) were identified as putative candidate genes. After performing the BOA approach, the 10% of F2 animals with the lowest breeding values for ΔRT were classified as low-ΔRT, and the 10% with the highest breeding values for ΔRT were classified as high-ΔRT. On average, 49.4% of low-ΔRT animals had 2 alleles from the Holstein breed (HH), and 39% had both alleles from the Gir breed (GG). In high-ΔRT animals, the average proportion of animals for HH and GG were 1.4 and 50.2%, respectively. This study allowed the identification of candidate genes for ΔRT in Gir × Holstein crossbred animals. According to the BOA approach, Holstein breed alleles could be associated with better response to heat stress effects, which could be explained by the fact that Holstein animals are more affected by heat stress than Gir animals and thus require a genetic architecture to defend the body from the deleterious effects of heat stress. Future studies can provide further knowledge to uncover the genetic architecture underlying heat stress in crossbred cattle.

Genome-wide association studies for tick resistance in Bos taurus × Bos indicus crossbred cattle: A deeper look into this intricate mechanism.

Rhipicephalus (Boophilus) microplus is the main cattle ectoparasite in tropical areas. Gir × Holstein crossbred cows are well adapted to different production systems in Brazil. In this context, we performed genome-wide association study (GWAS) and post-GWAS analyses for R. microplus resistance in an experimental Gir × Holstein F2 population. Single nucleotide polymorphisms (SNP) identified in GWAS were used to build gene networks and to investigate the breed of origin for its alleles. Tick artificial infestations were performed during the dry and rainy seasons. Illumina BovineSNP50 BeadChip (Illumina Inc., San Diego, CA) and single-step BLUP procedure was used for GWAS. Post-GWAS analyses were performed by gene ontology terms enrichment and gene transcription factors networks, generated from enriched transcription factors, identified from the promoter sequences of selected gene sets. The genetic origin of marker alleles in the F2 population was assigned using the breed of origin of alleles approach. Heritability estimates for tick counts were 0.40 ± 0.11 in the rainy season and 0.54 ± 0.11 in the dry season. The top ten 0.5-Mbp windows with the highest percentage of genetic variance explained by SNP markers were found in chromosomes 10 and 23 for both the dry and rainy seasons. Gene network analyses allowed the identification of genes involved with biological processes relevant to immune system functions (TREM1, TREM2, and CD83). Gene-transcription factors network allowed the identification of genes involved with immune functions (MYO5A, TREML1, and PRSS16). In resistant animals, the average proportion of animals showing significant SNPs with paternal and maternal alleles originated from Gir breed was 44.8% whereas the proportion of animals with both paternal and maternal alleles originated from Holstein breed was 11.3%. Susceptible animals showing both paternal and maternal alleles originated from Holstein breed represented 44.6% on average, whereas both paternal and maternal alleles originated from Gir breed animals represented 9.3%. This study allowed us to identify candidate genes for tick resistance in Gir × Holstein crossbreds in both rainy and dry seasons. According to the origin of alleles analysis, we found that most animals classified as resistant showed 2 alleles from Gir breed, while the susceptible ones showed alleles from Holstein. Based on these results, the identified genes may be thoroughly investigated in additional experiments aiming to validate their effects on tick resistance phenotype in cattle.

Post implantation development reveals that biopsy procedure can segregate "healthy" from "unhealthy" bovine embryos and prevent miscarriages.

Embryo biopsy has been performed in bovine in vivo produced embryos for the last twenty years, but little could be done with few embryonic cells in the past. Recently, advances in single cell analysis enabled a wide range of applications using embryo biopsy, from morphology to genetics analysis and different omics-techniques, which are promising for in vitro-fertilized (IVF) embryos. The aim of this study was to address if biopsy procedure would affect post implantation development of IVF blastocyts. Here we show that blastocyst stage do not affect re-expansion of biopsied embryos (regular blastocyst: 73.7%; expanded blastocyst: 73.1%), but affects (p<0.05) implantation (regular blastocyst: 37.8%, expanded blastocyst: 61.0%), so ideally biopsy should be performed in expanded blastocysts. No detrimental effect of biopsy procedure was detected for post-implantation development (calving rates, Biopsy: 47.1%, Control: 41.9%), and normal calves were born (Birth weight, Biopsy: 32.10±7.20kg; Control: 30.95±5.43kg). Surprisingly, we found interesting results suggesting embryo survival can be increased with aggressive procedures (such as embryo biopsy), and this is highly associated with early pregnancy loss (Biopsy: 0%, Control: 17.4%). This finding also suggests morphological classification of day 7 blastocysts is far from ideal, and supposedly, unhealthy embryos can implant but are bound to miscarriage during the first trimester (non-biopsied embryos). Our results show biopsy procedure is safe for bovine IVF embryos, and shed new light into the importance of conceptus in early pregnancy loss in cattle.

Genome wide scan for quantitative trait loci affecting tick resistance in cattle (Bos taurus x Bos indicus).

BACKGROUND: In tropical countries, losses caused by bovine tick Rhipicephalus (Boophilus) microplus infestation have a tremendous economic impact on cattle production systems. Genetic variation between Bos taurus and Bos indicus to tick resistance and molecular biology tools might allow for the identification of molecular markers linked to resistance traits that could be used as an auxiliary tool in selection programs. The objective of this work was to identify QTL associated with tick resistance/susceptibility in a bovine F2 population derived from the Gyr (Bos indicus) x Holstein (Bos taurus) cross. RESULTS: Through a whole genome scan with microsatellite markers, we were able to map six genomic regions associated with bovine tick resistance. For most QTL, we have found that depending on the tick evaluation season (dry and rainy) different sets of genes could be involved in the resistance mechanism. We identified dry season specific QTL on BTA 2 and 10, rainy season specific QTL on BTA 5, 11 and 27. We also found a highly significant genome wide QTL for both dry and rainy seasons in the central region of BTA 23. CONCLUSIONS: The experimental F2 population derived from Gyr x Holstein cross successfully allowed the identification of six highly significant QTL associated with tick resistance in cattle. QTL located on BTA 23 might be related with the bovine histocompatibility complex. Further investigation of these QTL will help to isolate candidate genes involved with tick resistance in cattle.

Efeito da aplicação de um análogo de GnRH (Buserelina) na concentração sérica de progesterona e na taxa de gestação em receptoras de embriões bovinos

O objetivo deste trabalho foi avaliar a ação de um análogo de GnRH (Buserelina), aplicado no dia da inovulação de embriões;nos níveis plasmáticos de progesterona e, conseqüentemente, na taxa de gestação de receptoras mestiças Holandês vsZebu. Foram realizadas cinco coletas de sangue a partir do dia da inovulação e intervaladas de 48 horas, para avaliar aconcentração de progesterona. Após 50 dias da inovulação, através do exame transretal, foi observado que 60% (12/20) dosanimais do grupo tratado ficaram gestantes e no grupo controle foi obtida uma taxa de gestação de 50% (10/20). Essesrestlttados não foram significativos pelo teste de Qui-quadrado. No entanto, os níveis séricos de progesterona dos animais dogrupo tratado foram maiores (p 0,05) que do grupo controle. Através dos resultados obtidos conclui-se que a aplicação de1 Omg de buserelina no dia da inovulação do embrião causou um incremento na concentração de progesterona dos animaistratados, sem contudo resultar em aumento na taxa de· gestação.