Characterization of follicle and CL development in beef heifers using high resolution three-dimensional ultrasonography.

The aim was to characterize dominant follicle (DF) and CL development through the estrous cycle of cattle using three-dimensional (3D) ultrasonography while making a comparison with conventional two-dimensional (2D) B-mode ultrasound (US) and to relate the measures taken to systemic concentrations of steroid hormones and gonadotropins. After synchronization of estrus, the ovaries of crossbred beef heifers (N = 5) were assessed using daily US with a GE Voluson i US scanner until the end of the first follicle wave, then every other day until emergence of the final (ovulatory) wave, when daily US resumed until ovulation. Follicle and CL growth were recorded and mapped. Measures of diameter (2D) and volume (3D) of the DF from the first and ovulatory waves of the cycles; and CL development were captured and stored for further analysis. Blood flow to the DF and CL were assessed using 3D power Doppler US measuring vascularization index (VI; %), vascularization flow index (0/100) and flow index (0/100). Jugular blood samples were collected every 24 hours for progesterone from the first estrus until the second ovulation. Concentrations of estradiol (E2) and follicle stimulating hormone (FSH) were measured every 8 hours from estrus to second follicle wave emergence; then, E2 only was measured from final follicle wave emergence until ovulation. Data were analyzed using PROC MIXED and PROC REG in SAS. Dominant follicle blood flow tended to decrease during follicle wave emergence and DF VI increased (P < 0.05) 24 hours before ovulation after peak E2. Measures of the DF and CL volume (3D) were highly predictive of 2D diameter measures throughout the cycle (P < 0.0001). Predictive values (r(2)) for day of wave emergence and day from ovulation were similar for 2D and 3D measures; however, 2D measures had higher repeatability when compared with 3D measures. There was no relationship between CL VI and progesterone early in the cycle (r(2) = 0.12; P = 0.1); however, there was a strong positive relationship approaching ovulation (r(2) = 0.77; P < 0.0001). In conclusion, 3D power Doppler measures of blood flow appears to be representative of vascular changes in the DF and CL throughout the estrous cycle. However, the extra time required to acquire and analyze a 3D image and the relatively little additional information obtained over that achievable with 2D imaging in terms of follicle and CL development might preclude its widespread use other than for detailed research purposes.

Effect of site of deposition on the fertility of sheep inseminated with frozen-thawed semen.

The widespread use of artificial insemination (AI) in sheep is currently prevented due to the lack of a cost effective insemination technique utilising frozen-thawed semen. The objective of the present study was to determine if the deposition of frozen-thawed semen in the vaginal fornix would result in a pregnancy rate comparable to that achieved following cervical insemination. Multiparous ewes of various breeds were synchronised and inseminated into either the vaginal fornix (n=78) or the cervix (n=79), at 57 h post sponge removal, with frozen-thawed semen. Information on mucus secretion and the depth to which it was possible to penetrate the cervix at insemination (cervically inseminated ewes only) was recorded at the time of AI. Pregnancy rate was subsequently determined either by return to service (oestrus) or after slaughter 30 days post insemination. Insemination site did not significantly influence pregnancy rate using frozen-thawed semen (36.2% compared to 27.6% for cervical and vaginal fornix insemination, respectively; P=0.26). Whilst depth of cervical penetration was positively associated with pregnancy rate (P<0.05), this association needs to be interpreted with caution as none of the ewes where the cervix could not be penetrated (score=0) was pregnant. In conclusion, pregnancy rate following insemination of frozen-thawed semen into the vaginal fornix was within 10% points of that obtained following cervical AI of frozen-thawed semen. As insemination into the vaginal fornix is technically easier than cervical insemination, it may be more practical for use in large scale applications.

Proteomic characterization of histotroph during the preimplantation phase of the estrous cycle in cattle.

Uterine secretions, or histotroph, are a critical component for early embryo survival, functioning as the sole supply of vitamins, minerals, enzymes, and other myriad of nutrients required by the developing conceptus before implantation. Histotroph is therefore a promising source for biomarkers of uterine function and for enhancing our understanding of the environment supporting early embryo development and survival. Utilizing label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) shotgun proteomics, we characterized the uterine proteome at two key preimplantation stages of the estrous cycle in high fertility cattle. We identified 300 proteins on Day 7 and 510 proteins on Day 13 including 281 proteins shared between days. Five proteins were more abundant (P < 0.05) on Day 7 compared with Day 13 and included novel histotroph proteins cytokeratin 10 and stathmin. Twenty-nine proteins were more abundant (P < 0.05) including 13 unique on Day 13 compared with Day 7 and included previously identified legumain, metalloprotease inhibitor-2, and novel histotroph proteins chromogranin A and pyridoxal kinase. Functional analysis of the 34 differentially expressed proteins (including 14 novel to histotroph) revealed distinct biological roles putatively involved in early pregnancy, including remodelling of the uterine environment in preparation for implantation; nutrient metabolism; embryo growth, development and protection; maintenance of uterine health; and maternal immune modulation. This study is the first reported LC-MS/MS based global proteomic characterization of the uterine environment in any domesticated species before implantation and provides novel information on the temporal alterations in histotroph composition during critical stages for early embryo development and uterine function during the early establishment of pregnancy.

Effects of lipid-encapsulated conjugated linoleic acid supplementation on milk production, bioenergetic status and indicators of reproductive performance in lactating dairy cows.

Conjugated linoleic acid (CLA) reduces mammary milk fat synthesis in a dose-dependent manner. Our objective was to determine the effects of lipid-encapsulated CLA (LE-CLA) supplementation on milk production, reproductive performance and metabolic responses in lactating dairy cows fed a grass silage-based diet. Seventy-two Holstein-Friesian cows (32 primiparous and 40 multiparous) were used in a completely randomized block design. Cows received either 80 g of LE-CLA daily or 60 g of calcium salts of palm fatty acids daily (control) from parturition until 60 days in milk. LE-CLA contained a 50:50 mix of cis-9,trans-11 CLA and trans-10,cis-12 CLA, resulting in a daily intake of 6 g of each isomer. Milk production and dry matter intake were recorded daily, and blood samples were collected 3-times a week. Blood samples were analysed for circulating concentrations of glucose, non-esterified fatty acids (NEFA), ß-hydroxybutyrate (BHBA), insulin and insulin-like growth factor-I (IGF-I). Progesterone was measured in blood samples collected after the first post-partum insemination. Ovarian ultrasound examinations commenced at 8-10 d post partum and were carried out 3-times a week until first ovulation. LE-CLA treatment resulted in decreased milk fat concentration, with consequent improvements in energy balance and body condition score (BCS). The peak concentration of NEFA in blood was reduced by LE-CLA, but circulating concentrations of insulin, glucose, IGF-I, BHBA and progesterone were not affected. There was no effect of LE-CLA supplementation on the post-partum interval to first ovulation. Services per conception tended to be reduced. The reduction in milk energy output and improvement in energy status and BCS in LE-CLA-supplemented cows provides a strong rationale for further studies with greater cow numbers to test effects on reproductive performance.

Transmission ratio distortion at the growth hormone gene (GH1) in bovine preimplantation embryos: An in vitro culture-induced phenomenon?

The growth hormone gene (GH1) and its polypeptide product (GH) have a crucial role in reproduction, embryogenesis and general development. A polymorphism present in the fifth exon of the bovine GH1 gene (GH1 p.Leu127Val) has been associated with GH release and milk production in cattle. The objective of the present study was to examine the genotype frequencies of the GH1 p.Leu127Val polymorphism in bovine blastocysts produced in vitro and in vivo to determine if allelic variation of the GH1 gene affects embryo development and survival. A heterozygous (p.Leu127/Val127) sire was used for in vitro fertilization of oocytes of unknown maternal genotype (n = 104) and known maternal genotype (n = 115). PCR amplification and genotyping of the GH1 gene from Day 8 blastocysts derived from these fertilized oocytes demonstrated that there was significant over-representation from the expected Mendelian ratio of GH1 p.Leu127/Leu127 homozygotes from oocytes of known maternal genotype (P = 0.006). Contrary to this, analysis of in vivo-produced bovine blastocysts of known parental GH1 genotype (n = 69) did not reveal an overrepresentation of GH1 p.Leu127/Leu127 homozygotes. These results suggest that developing in vitro-produced embryos are exposed to a selection process, probably due to a less favorable culture environment, that acts to increase the number of GH1 p.Leu127/Leu127 homozygotes, thereby giving rise to the observed transmission ratio distortion (TRD) of GH1 genotypes when compared to in vivo produced embryos.

Temporal expression of transcripts related to embryo quality in bovine embryos cultured from the two-cell to blastocyst stage in vitro or in vivo.

The post-fertilization embryo culture environment can have a dramatic effect on the pattern of gene expression in the embryo and it is widely acknowledged that bovine embryos derived from in vitro culture are of inferior quality to those derived in vivo. The objective of this study was to examine temporal variation in the mRNA abundance of several transcription and translation factors known to differ between blastocysts produced following culture in vitro and in vivo. Embryos were recovered from two in vitro culture systems SOF1 or SOF2 at five developmental stages: 2- to 4-cell, 8-cell, 16-cell, morula, and blastocyst. In vivo embryos were produced from superovulated and artificially inseminated heifers and recovered at approximately 40 hr or 3, 4, 5, and 7 days postinsemination. Blastocysts were also produced following in vitro maturation, in vitro fertilization and culture in the ewe oviduct. Analysis of relative transcript abundance for FOXO3A, EEF1G, HMG2, and REA was performed using quantitative real-time PCR. Irrespective of culture environment each transcript followed, approximately the same general pattern of expression where relative abundance decreased dramatically from the 2- to 4-cell stage to 8-cell stage and increased from the morula to blastocyst stage (P < 0.05). Transcripts for GNBL2 were not observed between the 2- and 16-cell stage of development. Relatively high expression at the 2- to 4-cell indicated that these transcripts are most likely of maternal origin produced in the oocyte during growth and final maturation. A culture-induced change in mRNA abundance of transcription and translation factors was evident in embryos that were produced not only between in vivo and in vitro culture environments but also between different in vitro culture systems.

Species-related differences in blastocyst quality are associated with differences in relative mRNA transcription.

The objective of this study was to compare the relative transcript abundance of several important candidate genes between ovine and bovine blastocysts. Blastocysts were produced by in vitro maturation, fertilization, and subsequent culture in one of two formulations of synthetic oviduct fluid medium (SOF1 and SOF2). From each IVF replicate groups of 10 bovine and 10 ovine blastocysts from each of the two media were used for analysis of mRNA relative abundance. Transcript levels for mitochondrial Mn-superoxide dismutase (MnSOD), survivin, and glucose transport 5 (Glut-5) were significantly higher in ovine blastocysts than bovine (P < 0.05), while transcripts for Connexin 31 (Cx31), interferon tau (IFN-tau), and sarcosine oxidase (SOX) were significantly more abundant in bovine blastocysts (P < 0.01). For the two remaining transcripts, E-cadherin (E-cad) and Na/K ATPase (Na/K), there was no difference. Culture of bovine embryos in SOF2 resulted in a significant increase in the level of expression of MnSOD and Glut-5 (P < 0.05) compared to those bovine embryos cultured in SOF1. For all the other transcripts, except survivin, there was a significant decrease in the relative abundance. Culture of sheep embryos in either SOF1 or SOF2 did not have a major influence on transcript abundance; of the eight transcripts examined, the relative abundance of only one, SOX, was significantly altered. Bovine blastocysts produced in SOF2 had significantly higher survival rates at 24, 48, and 72 hr and significantly higher hatching rates following vitrification and warming than those cultured in SOF1 (P < 0.001). In conclusion, we have quantified for the first time the mRNA expression of a set of important developmental genes in sheep blastocysts and we have demonstrated that these differences between species in their adaptability to culture conditions, manifested in differences in embryo morphology and cryotolerance, are related to differences in mRNA relative abundance. The results also highlight the usefulness of transcript analysis as a marker of embryo quality.