Post Insemination Research Articles

A Comparative Study on Certain Blood Metabolites and Hormonal Indices for Detection of Pregnancy of Crossbred Cows of High Altitude: Mizoram

Background: Early detection of pregnancy can perk up the reproductive competence of dairy animals and decreasing the interval between two successive AI. Several methods of pregnancy diagnosis both direct and indirect are being practiced; but to date, none of these technologies qualifies as the ideal pregnancy detection method due to its limitation. The present study was designed to learn about the changes of hormonal and blood metabolites during early bovine pregnancy. Methods: A total of 30 numbers of crossbred cows at 2nd to 5th lactation was selected for the study. Clinico-gynaecological examination was carried out in all the cows and those have devoid of ill health, having a regular estrous cycle and estrus period were inseminated at mid to late estrus. Ten milliliter of blood samples were collected aseptically from all the cows (6 samples from each cow and a total of 180 samples) on day 1, 10, 20, 30, 60 and 90 of post-A.I. From the obtained serum, levels of glucose, cholesterol, BUN, total protein and hormonal profile estrone sulphate, progesterone were analysed. All the cows were observed for the absence of external signs of estrus after AI and pregnancy was confirmed by using ultrasonography and per rectal examination on day 30 to 40 of post insemination. Result: It was observed that level of glucose, BUN, cholesterol and progesterone was significantly increased during early pregnancy (P less than 0.01). While, the serum level of estrone sulphate significantly decreased while advancing the pregnancy. There was positive correlation between the level of progesterone (P less than 0.01) and negative correlation of estrone sulphate (P less than 0.05) with serum glucose, BUN, cholesterol at different days of post A.I. The blood biochemical constituents i.e. glucose, BUN, cholesterol and hormone estrone sulphate, progesterone possibly will used as an ideal marker of early pregnancy in cattle. Thus blood biochemical constituents like glucose, BUN, cholesterol and hormone estrone sulphate, progesterone possibly can be used as a tool for diagnosis of non pregnant dairy cattles with very high accuracy at around 30 days of post insemination.

Comparative ejaculatory response, fresh and frozen semen quality and fertility to artificial vagina vs electroejaculation method of semen collection in mithun (Bos frontalis) bulls.

The study aimed to describe a comparative ejaculatory response, fresh and frozen-thawed semen quality and fertility of semen collected by artificial vagina and electroejaculation in mithun. Experimental bulls were divided into two groups, G-I: young bulls (n=4; 4-5years of age) and G-II: older bulls (n=4; 8-10years of age). Sixteen ejaculates were collected from each group G-I (AV1 ) and G-II (AV2 ) by artificial vagina method (control). Thirty-six ejaculates were collected from the same bulls from each group G-I (EE1 ) and G-II (EE2 ) by electroejaculation method (treatment). The study did not reveal any significant (p>0.05) difference in the ejaculatory responses between EE1 and EE2 . Mann-Whitney U test indicated that salivation discomfort sign score was significantly (p<0.05) higher in EE1 . Fresh and frozen-thawed semen quality parameters, and motility and velocity profiles recorded by computer-assisted sperm analyser were significantly (p<0.05) lower in electroejaculation than the artificial vagina. The conception rates (AV1 vs EE1 & AV2 vs EE2 ) at day 35-45 post insemination were nonsignificantly higher (p>0.05) in the artificial vagina group. It concluded that, although artificial vagina method has better semen quality, nevertheless, electroejaculation has the potential for semen collection from free-range mithun bulls to incorporate in assisted reproductive technology procedures.

The Effects of Heat Shock Protein 70 Addition in the Culture Medium on the Development and Quality of In Vitro Produced Heat Shocked Bovine Embryos.

Simple SummaryEuropean dairy cows are extremely sensitive to heat stress. During hot summers milk yield is suppressed, welfare status of the cows is aggravated, and their fertility is seriously compromised. The observed subfertility is attributed to endocrinological alterations and mainly to inability of the early embryo to develop uneventfully until the implantation stages. To protect the integrity and the functionality of their cells, all mammals have inherent defense mechanisms against external insults. A major representative of these molecules are the heat shock proteins (HSPs) that are secreted by almost all cells. In this study, we examined the effects of exogenous HSP70 on the developmental competence and quality of early embryos produced in vitro, after 24-h exposure to slightly elevated temperature. Our findings imply that the temperature rise is detrimental for the developmental potential of the early embryos, and that the HSP70 can partly mitigate the harmful effects of heat stress, by improving the embryo yield and by reducing the adverse effects of heat stress on some embryo quality characteristics.The aims of the present study were to examine the effects of HSP70 addition in the in vitro culture medium of day 3 embryos on their developmental competence and quality. Bovine oocytes (n = 1442) were in vitro matured, inseminated and cultured for the first two days according to standardized methods. The presumptive zygotes were randomly allocated in three experimental groups: Control, C (embryos cultured at 39 °C throughout the culture period), group C41 (temperature was raised to 41 °C from the 48th to 72nd h post insemination (p.i.) and then it returned at 39 °C for the remaining culture period), and group H41 (the temperature modification was the same as in C41 and during heat exposure, HSP70 was added in the culture medium). Cleavage and embryo yield were assessed 48 h p.i. and on days 7, 8, 9, respectively and gene expression in day 7 blastocysts was assessed by RT-PCR. Blastocyst yield was the highest in group C39; and higher in group H41 compared to group C41. From the gene expression analyses, altered expression of 11 genes was detected among groups. The analysis of the orchestrated patterns of gene expression differed between groups. The results of this study confirm the devastating effects of heat stress on embryo development and provide evidence that HSP70 addition at the critical stages can partly counterbalance, without neutralizing, the negative effects of the heat insult on embryos, acting mainly through mechanisms related to energy deployment.

Delayed Ovulation in Bovine: Diagnosis and Treatment

Repeated breeder dairy cows (96) were used in this study. They were diagnosed clinically as cases of delayed ovulation and divided into five groups according to type of treatment used post insemination. Group one which includes 28 cow (1=28) treated with 1 vial of fertagyl, (2=22) treated with 2 vials of fertagyl, (3=19) treated with 5 ml of chorionic gonadotropin, (4=17) treated with 1 vial of Nymfalom and (5=10) treated with 1 vial of chorulon. Results showed that the number of cows which did not return to estrus (responded) were 16(57.14%), 12(54.54%), 9(47.47%), 12(70.6%) and 4(40%) respectively. Treatment and insemination were repeated to a number of unresponded cows and their results revealed response of 3 cows (27.27%) from group (1=11), response of 4 cows (50%) from group (2=8), response of 3 cows (42.85%) from group (3=7), response of 1 cow (20%) from group (4=5) and response of 2 (50%) from group (5=4). Data concerning the number, type, sex and viability of the offspring born from the treated cows were included in this study. Finally we may conclude that Nymfalon showed better result in treating cases of delayed ovulation and in improving their conception and pregnancy rate. The remaining treatments were also advisable in the absence of Nymfalon. The use of the same treatment again was also beneficial in some unresponded cases.

Expression profiling of chemokines CCL8 and CXCL10 during peri-implantation period in Murrah buffaloes as a possible signature of embryonic implantation

The present investigation was aimed to evaluate novel implication of chemokine genes CXCL10 (C-X-C motif chemokine 10) and CCL 8 (C-C motif chemokine 8) genes for use as ideal pregnancy biomarker in dairy buffaloes. We studied expression profile of both these chemokine genes in whole blood of Murrah buffaloes on day 12, 15, 18 and 21 post artificial insemination (AI) using SYBR green chemistry based quantitative real time reverse transcription PCR. Our investigations revealed a consistent increase in transcriptional abundance of CCL8 and CXCL10 genes during this period, although the difference in expression level was not significant between day 15 and 18 post AI for CXCL10 gene. We also studied the effect of parity on the expression profile of these two genes and found that expression level of both these genes is independent of parity status of the animal. Based on the results, it can be concluded that these chemokine genes can be used as early pregnancy biomarker on any day between days 12 to 21 post artificial insemination in buffaloes irrespective of their parity status.

Influence of Different Regimes of Moderate Maternal Feed Restriction during Pregnancy of Primiparous Rabbit Does on Long-Term Metabolic Energy Homeostasis, Productive Performance and Welfare.

Simple SummaryIn rabbit farms, the main production costs come directly from food supplies. Although the reproductive outcomes in this species are acceptable, the results are worse when it comes to primiparous rabbits, so it is recommended that insemination be carried out post-weaning. By avoiding the overlap of the second gestation and the first lactation, better fertility results are expected. Still, despite this, the rabbits garner more adipose tissue than desired, and the productive efficiency deteriorates in the long term. The purpose of this study was to evaluate the influence of different periods of moderate feed restriction (one, two or three weeks) applied during the second pregnancy of primiparous does. We studied fetoplacental development, productive parameters, metabolism and possible stress indicators. Results showed that the voluntary feed intake of dams increased right after feed restriction. No permanent alterations were found in reproductive outcome, metabolism or welfare of does, meaning that this feeding strategy could be successfully applied in rabbit farms.In this study, a maternal feed restriction (MFR; 105 g/d) in primiparous rabbit does was applied from day 0 to 7 post artificial insemination (AI) (R07, n = 96), from day 7 to 21 post AI (R721, n = 92), from day 0 to 21 post AI (R021, n = 94) or fed ad libitum during whole pregnancy (Control, n= 92). Feed intake (FI) was measured after MFR was over. On day 28 of gestation, fetoplacental development was evaluated (n = 11/group) and the productive parameters of the remaining dams were analyzed. Plasma free tri-iodothyronine (T3) and thyroxine, glucose, insulin, non-esterified fatty acids (NEFA), and corticosterone were analyzed during gestation and lactation (n = 5/group). After MFR, all groups significantly increased their voluntary FI. The longer MFR was, the lower the weight and length of the fetuses, but no long-term effects over litter performance were observed. R021 groups had the lowest T3 and the highest NEFA concentrations during pregnancy and showed insulin resistance at the end of gestation, but during lactation, energy homeostasis was balanced in all groups. MFR did not affect corticosterone concentrations. In conclusion, the ration setting applied slightly involved the energy homeostasis and metabolism of the animals, but their overall metabolic condition, productive performance and welfare were not compromised.

Influence of breed on the quality of in vivo produced embryos from Boran and Holstein Friesian cross dairy breed in Ethiopia

The variation of the dairy breed can determine the success of bovine embryo transfer by influencing the quantity and quality of in vivo embryo production. In this experiment, output and quality of in vivo produced embryos using semen of progeny tested Holstein Friesian (HF) sire in Boran and HF*Boran F1 cross cows, and semen from purebred Boran sire in HF*Boran F1 cross and Boran cows were evaluated. Boran (n=18) and HF*Boran cross (n=18) breed donor dams were superovulated using a previously optimized follicular stimulating hormone (FSH) (Pluset®) dose regimen: 650 IU for HF*Boran cross and 250 IU for Boran breeds. Each cow was flushed on Day-7 post insemination and embryos were evaluated for their developmental stages and quality. Superovulatory response rates were 88.9% and 83.3%, respectively, for Boran and HF*Boran with no significant (P&gt;0.05) breed differences. Total recovery rates were relatively lower (56.5%) in Boran compared to in HF*Boran (67.4%). The mean (±SE) embryo flush outputs were 6.5±0.8 for Boran and 6.9±0.7 forHF*Boran with no significant breed difference. Recovery of a transferrable embryo was significantly higher (68.0%; P&lt;0.05) in HF*Boran dam inseminated with HF sire semen. Boran cows yielded a significantly higher (P&lt;0.05) proportion of unfertilized ovum (57.6 %) irrespective of the sire breeds. Comparatively, a higher number of degenerated embryos were produced by HF*Boran cows. This study demonstrated that the presence of breed-related differences in both the quality and quantity of in vivo produced Bovine embryos.

P–171 Automated oocyte and zygote denudation using a novel microfluidic device supervised by a computer vision algorithm

Abstract Study question Is it possible to remove cumulus cells using a 16-well microfluidic device with automated flows to facilitate vitrification, ICSI, NI-PGT or non-invasive metabolomics analysis? Summary answer The designed automated system and protocol efficiently denude 16 samples simultaneously with a x10 lower shear stress than the manual process and without human intervention. What is known already Most processes involved in IVF such as insemination, washing, denudation, embryo culture and selection are still manually performed, labor-intensive and require highly skilled professionals. This leads to a significant variability in the clinical outcomes achieved by different embryologists and labs. The automation of these processes is a promising approach to reduce costs and improve the accessibility to assisted reproductive therapies. Although a simple procedure, standardization of cumulus oocyte complex (COCs) and zygotes denudation is key to facilitate ICSI, vitrification and to avoid DNA contamination for NI-embryo testing (PGT or metabolomics), while avoiding damage to the oocyte by excessive shear stress. Study design, size, duration A total of 160 cow COCs were used due to their size similarity with human COCs. Half were denuded 16–20 hours post-insemination and half pre-insemination for 5–10 minutes. COCs were classified as partially denuded if fertilization assessment, ICSI or vitrification was possible, and completely denuded if no cumulus cells remained. COCs controls were manually denuded (Stripper® capillary 145μm ID) to compare shear stress between procedures. This study was conducted during 2020 – 2021. Participants/materials, setting, methods We developed a customized microfluidic biochip that exerts a particular fluid motion while avoiding egg entrapment within microfluidic channels. The denudation efficacy was established by subjectively scoring images of bovine oocytes after generating a continuous “Push &amp; Pull” fluid motion inside the biochip wells. A Computer Vision model was developed in parallel in order to optically assess denudation completion. The model used was a Pytorch implementation of Faster-RCNN with ImageNet pretrained weights Main results and the role of chance 96 bovine COCs were microfuidically handled post insemination achieving complete (56/96) or partial (40/96) removal of the cumulus cells on day 1, while for day 3 double denudation group, 89/96 (92.7%) were completely denuded while the rest remained partially denuded. In comparison, 80/80 (100%) of manually denuded cow COCs, achieved complete denudation (50% post-insemination group and 50% pre-insemination group). In addition, 48/64 (75%) cow COCs treated pre-insemination were partially denuded, enough to carry out ICSI after 5–10 min of treatment. The results here obtained indicate that media needs to flow through the device at a rate that can generate enough shear to strip off the cumulus-corona cells while avoiding emptying of the reservoirs containing the fertilization or culture medium. The shear stress of our design was calculated to be smaller than 4.4 Pa, about ten times lower than the one applied by the manual process (∼44Pa). The deep learning algorithm was tested on 20 unseen human oocytes on day 1, with 10 true positives 9 true negatives, and 1 false negative (95% accuracy). Limitations, reasons for caution The success of the denudation procedure was dependent on the design of the biochip wells and the microfluidic protocol used. The accuracy of our findings is still limited because of the difficulty in manufacturing prototype biochips. Wider implications of the findings: Complete denudation is key to avoid DNA contamination for NI-PGT or metabolomics analysis, while avoiding damage to the oocyte by excessive shear stress. Our device, which has the potential of scaling up and treat each oocyte individually, can improve automation and increase efficiency of current ART procedures Trial registration number NA

P–206 Does oocyte vitrifcation affect morphokinetics of subsequent embryo development?

Abstract Study question Are the morphokinetic profiles, as assessed using time-lapse technology, of human embryos developed from vitrified oocytes different to those from fresh oocytes. Summary answer Vitrification of oocytes does have an effect on early developmental morphokinetic profiles, but this is normalized by the time the embryo has reached blastocyst. What is known already Vitrification of oocytes is now commonplace, but little is known about the effect this may have on subsequent embryo development. Study design, size, duration This was a retrospective data analysis, from 8 fertility clinics in the UK between 2012 and 2019. Embryos from patients in the vitrified group (n = 557) were matched to fresh patient controls (n = 539). The matching was performed based on the following criteria: type of treatment, patient age, cause of infertility and number of embryos. Participants/materials, setting, methods The embryos in each group were compared for mean morphokinetics of key developmental stages in hours post insemination (hpi). Parameters compared included early cleavage divisions (t2-t8), time to start of compaction (tSC), time to morula (tM), time to start blastulation (tSB), time to full blastocyst (tB) and duration of compaction (tB-tSC). Treatment outcomes were compared between the two groups, including percentage of blastocyst formation, clinical pregnancy rate, implantation rate and live birth rate. Main results and the role of chance The results showed a significant delay across all early cleavage divisions as follows for vitrified and fresh oocytes respectively: 2-cell (28.14 vs 26.10 (p &amp;lt; 0.001)), 3 cell (37.56 vs 35.37 (p &amp;lt; 0.001)), 4 cell (40.58 vs 37.54 (p &amp;lt; 0.001)), 5 cell (50.31 vs 47.14 (p &amp;lt; 0.001)), 6 cell (53.99 vs 50.87 (p &amp;lt; 0.001)), 7 cell (57.08 vs 54.48 (p &amp;lt; 0.001)) and 8 cell (61.26 vs 58.91 (p &amp;lt; 0.01)). In addition, tSC was also significantly delayed in the vitrified group (80.65 vs 76.36 (p &amp;lt; 0.001)). However, the compaction stage was significantly shorter in the vitrified oocytes (19.02 vs 22.45 (p &amp;lt; 0.001)). Therefore, there was no difference in the time that embryos derived from fresh and vitrified oocytes reached the blastocyst stage (108.03 vs 107.78 (p &amp;gt; 0.05)). No difference was found in clinical pregnancy, implantation or live birth rates but significantly fewer blastocyst developed from vitrified oocytes compared to fresh (36.09% vs 42.4% (p &amp;lt; 0.05)). Limitations, reasons for caution Although this was a matched analysis, it was a retrospective in nature therefore is subject to confounders. However, it would be problematic to perform a prospective randomized controlled trial to address this study question given the need to randomize patients to elective freezing of oocytes prior to embryo creation. Wider implications of the findings: Vitrification of oocytes may affect early developmental morphokinetic profiles, but any effect is normalized by the time the embryo has reached blastocyst. However, fewer blastocysts may develop following oocyte vitrification. This may have implications for oocyte donation banks and those patients choosing to cryopreserve oocytes. Trial registration number NA

P–179 Timing of blastocyst observation on day 5: effect on the assessment to predict live birth, and the incorporation into a blastocyst selection model

Abstract Study question Does variation in day 5 observation timing confound embryo-morphology-based live birth prediction, and is it possible to develop a robust comprehensive numerical prediction model. Summary answer Day 5 observation timing confounds embryo-morphology-based live birth prediction. A robust comprehensive numerical prediction model can be developed after considering a number of contributing variables. What is known already Embryo development is a dynamic process, and therefore the widely used static observations potentially lead to biased prediction of live birth outcomes. So far, little is known in regard to potential confounding impact of day 5 assessment timing on the static-morphology-based live birth prediction. In addition, the inter-observer variation in morphology-based embryo assessment requires a more robust system to improve consistency of selection. Study design, size, duration This retrospective multi-center cohort study included 8866 autologous oocyte in vitro fertilisation treatment cycles performed at 14 associated clinics within the same network during 2012–2018. Only fresh cycles with single day 5 embryo transfers were included for analysis with all pregnancies followed up until birth. Repeat cycles of same patients were excluded to avoid clustering effect in statistical analysis. Participants/materials, setting, methods Dataset was randomly split into two subsets at 60:40 ratio, with one (n = 5274) used for regression analysis and model development and the other (n = 3592) used for model testing. Multiple logistic regression was performed to evaluate live birth predicting power of several potential contributors, expressed by odds ratio (OR) and 95% confidence interval (CI). A comprehensive prediction model was subsequently developed based on calculated weights of contributing factors, then tested via receiver operating characteristics (ROC) analysis. Main results and the role of chance The timings of day 5 observation of 8866 included embryos, measured by hours post insemination (HPI), distributed in a bell shape ranging from 112.0 to 120.0 h (mean±SD 115.7±1.7 h). After taking into account female age at egg collection (grouped as &amp;lt; 30 yr, 30–34 yr, 35–39 yr, 40–44 yr, and 45 yr or older), whether or not the first egg collection, number of eggs collected, embryo developmental stage (grouped as pre-blastocyst, early blastocyst, expanding blastocyst, expanded blastocyst, and hatching/hatched blastocyst) and morphology score(A/B/C/D); multivariate logistic regression analysis showed significant association (OR 1.096, 95% CI 1.020–1.177, P = 0.012) between HPI groups (112–113.9 h, 114–115.9 h, 116–117.9 , and 118–120 h) and subsequent live birth outcomes. A comprehensive numerical scoring system was developed based on the statistically significant predictors including female age (OR 1.465, 95% CI 1.364–1.574, P = 0.000), embryo developmental stage (OR 1.341, 95% CI 1.244–1.445, P = 0.000), morphology score (OR 1.520, 95% CI 1.392–1.661, P = 0.000) and HPI (OR mentioned above); with a formula of Score = (Female_age_group/5)*1.465 + (Developmental_stage/5)*1.341 + (Morpho_Score/4)*1.520 + (HPI_Group/4)*1.096. ROC analysis showed statistically significant predictive power of the resulting model as expressed by area under the ROC curve using both the development (0.690, 0.675–0.704, P = 0.000) and testing (0.685, 0.667–0.703, P = 0.000) subsets. Limitations, reasons for caution The retrospective design does not allow for controlling of unknown confounders. HPI was based on static observations in this study so future time-lapse study may bring more insights with more accurate observation and measurement. Wider implications of the findings: The varying HPIs at day 5 observation were alarming as this could confound live birth prediction using embryology parameters. It is important to standardise the timing of embryo observations. The inclusion of HPI into a comprehensive numerical scoring system for live birth prediction may potentially improve its robustness Trial registration number Not applicable

Heat Shock Protein 70 Improves In Vitro Embryo Yield and Quality from Heat Stressed Bovine Oocytes.

Simple SummaryThe Holstein cows are among the most thermosensitive farm animals. In this breed, during the heat stress periods, fertility is seriously compromised due to induced alterations of the endocrine status, reduced fertilizing capacity of the oocyte and increased embryo deaths. To combat the deleterious effects of stress, cells synthesize a series of specific molecules that are mainly involved in cellular protection against the heat insult, called heat shock proteins (HSPs). Here, we examined the effects of supplementing HSP70 in in vitro matured bovine oocytes under thermoneutral or heat stress conditions, and we assessed its efficacy on in vitro embryo yield and quality; the latter was determined on the basis of the expression of various genes related to important cellular functions. It was manifested that HSP70 addition into the in vitro maturation medium restores the developmental competence of heat stressed oocytes and improves the quality of the in vitro produced embryos.Heat shock protein 70 (HSP70) is a chaperon that stabilizes unfolded or partially folded proteins, preventing inappropriate inter- and intramolecular interactions. Here, we examined the developmental competence of in vitro matured oocytes exposed to heat stress with or without HSP70. Bovine oocytes were matured for 24 h at 39 °C without (group C39) or with HSP70 (group H39) and at 41 °C for the first 6 h, followed by 16 h at 39 °C with (group H41) or without HSP70 (group C41). After insemination, zygotes were cultured for 9 days at 39 °C. Cleavage and embryo yield were assessed 48 h post insemination and on days 7, 8, 9, respectively. Gene expression was assessed by RT-PCR in oocytes, cumulus cells and blastocysts. In C41, blastocysts formation rate was lower than in C39 and on day 9 it was lower than in H41. In oocytes, HSP70 enhanced the expression of three HSP genes regardless of incubation temperature. HSP70 at 39 °C led to tight coordination of gene expression in oocytes and blastocysts, but not in cumulus cells. Our results imply that HSP70, by preventing apoptosis, supporting signal transduction, and increasing antioxidant protection of the embryo, protects heat stressed maturing bovine oocyte and restores its developmental competence.

Setting the Cycle for Reproductive Success with a Pre-synchronization Program in Postpartum Beef Cows

Abstract Previous experiments from our laboratory determined that postpartum beef cows with a functional corpus luteum (CL) at the start of an estrous synchronization protocol have greater pregnancy success to FTAI than those that did not have a CL. Therefore, it was hypothesized that incorporation a presynchronization program prior to FTAI in postpartum cows will improve pregnancy rates. The goal of this project was to compare the effectiveness of a presynchronization program prior to the 7-d CO-Synch+CIDR fixed timed artificial insemination (FTAI) protocol in postpartum cows. Postpartum cows (n = 310) at two locations were randomly assigned to receive either the presynchronization program, consisting of 2 PGF2α injections 14 d apart and 11 d prior to the start of the 7-d CO-Synch+CIDR protocol, or the 7-d CO-Synch + CIDR protocol. Following estrus synchronization, all cows were inseminated by trained technicians using semen from bulls of known fertility. Pregnancy status to FTAI was assessed using ultrasonographic diagnosis at 30 d post insemination. Data were analyzed using a MIXED procedure of SAS and examined for effects of inseminator, body condition score (BCS), and location and age. Statistical significance was determined at P &amp;lt; 0.05 and a tendency at 0.05&amp;lt; P &amp;lt; 0.10. Age, BCS, and location did not affect (P &amp;gt; 0.05) pregnancy rates. Cows exposed to the pre-synchronization program tended to have greater AI pregnancy rates compared to cows that did not receive the pre-synchronization program (57.7±5.3 vs. 46.9±5.3%, respectively; P = 0.0687). AI pregnancy rates were greater in cows with &amp;gt;1 ng/mL P4 compared to cows with &amp;lt; 1 ng/mL P4 at the start of FTAI (77.7±7 vs. 55.7±6.2%, respectively; P = 0.0204). Furthermore, AI pregnancy rates were decreased by inseminator C (26.1±7.4) compared to the other inseminators this breeding season (A-54.4±7.9; B- 62.3±5.3; D-57.8±7.2; and E -51.1±5.2; P = 0.0026). Based on these data, incorporating a pre-synchronization program to ensure the presence of a CL at the start of a FTAI protocol may improve pregnancy success in postpartum beef cows.

PSV-8 Effects of Air Exposure and Agitation on Quality of Stored Boar Semen Samples

Abstract Recent adoption of post cervical artificial insemination facilitates lowered concentrations of semen inseminated. In order to achieve this reduced concentration, the volume of the insemination dose has been reduced and replaced with additional air in the semen storage container. The objective of this study was to investigate the effects of semen volume, air contact inside semen dose tubes, daily agitation of semen doses and extender type on semen quality, thermo-resistance and bacterial growth in extended boar semen doses over 7 days of liquid storage at 17 C. Ejaculates from 4 terminal cross-bred boars were collected for 4 weeks and used in the 3 x 2 x 2 factorial study. The effects of treatment (CON: 80ml doses sealed at the top of the tube; 40HIGH: 40ml doses sealed at top of tube; and 40LOW: 40ml doses sealed at top of the liquid), extender type (long-term vs short-term), and agitation (agitated vs not agitated) were investigated. The result of the study revealed that motility (P=0.014) and viability (P=0.007) in 40HIGH were lower than CON. pH (P&amp;lt; 0.001) was higher in 40HIGH compared to CON. Agitation did not impact motility (P=0.541), viability (P=0.406) or morphology (P=0.970) while long-term extender maintained higher motility (P=0.034), viability (P&amp;lt; 0.001) and normal acrosomes (P&amp;lt; 0.001) compared to short-term extender. VAP (P=0.039) of 40HIGH was lower than CON in a thermo-resistance test. Bacteria were cultured on both sheep’s blood and MacConkey agar and neither treatment (P=0.798; 0.766) nor agitation (P=0.396; 0.476) impacted bacterial growth in this study. In conclusion, semen doses prepared with 80mL or 40mL of total volume with minimal air contact in the tubes yield more desirable semen quality, as air contact negatively impacts boar semen pH and sperm motility. Additionally, regardless of volume or air exposure, daily agitation of boar semen doses did not affect semen quality.

Influence of parity and live weight on the concentration of Pregnancy-Specific Protein B (PSPB) in Kedah Kelantan (KK) cattle

The pregnancy-specific protein B (PSPB) is the protein of binucleated cells in the ruminant’s placenta. It contains several antigenic variants, which contributed to the ability to detect pregnancy from day 20 of pregnancy until parturition. In this research, the main objective is to determine the concentration of PSPB in serum and urine and their relation to Kedah-Kelantan (KK) cattle’s parity and live weight. Ten cattle (n=10) were selected for this study. The blood serum (2 ml) and urine samples were collected at days 0, 24, 42, and 164 post artificial insemination (AI). All the samples were centrifuged at 3000 rpm for 15 minutes at 4°C. The serum and urine were analyzed with competitive ELISA test kit and read at 450 nm wavelength speed. The standard OD value provided by the kit created the equation to calculate the concentration of PSPB in the serum and blood. Then, the correlation and multiple linear regression of parity and live weight were analyzed with a significant level of P < 0.01. Through the finding, the live weight and parity were positive correlate with PSPB in serum. The relationship between live weight and PSPB concentration in urine showed a negative correlation but there no correlation between parity and PSPB concentration in urine. The regression analysis result was 35% and 36% for PSPB concentration in serum and urine, respectively. This study suggests that cattle parity and live weight did not significantly affect the PSPB concentration.

Transmission of Bluetongue Virus Serotype 8 by Artificial Insemination with Frozen-Thawed Semen from Naturally Infected Bulls.

Transmission of bluetongue (BT) virus serotype 8 (BTV-8) via artificial insemination of contaminated frozen semen from naturally infected bulls was investigated in two independent experiments. Healthy, BT negative heifers were hormonally synchronized and artificially inseminated at oestrus. In total, six groups of three heifers received semen from four batches derived from three bulls naturally infected with BTV-8. Each experiment included one control heifer that was not inseminated and that remained BT negative throughout. BTV viraemia and seroconversion were determined in 8 out of 18 inseminated heifers, and BTV was isolated from five of these animals. These eight heifers only displayed mild clinical signs of BT, if any at all, but six of them experienced pregnancy loss between weeks four and eight of gestation, and five of them became BT PCR and antibody positive. The other two infected heifers gave birth at term to two healthy and BT negative calves. The BT viral load varied among the semen batches used and this had a significant impact on the infection rate, the time of onset of viraemia post artificial insemination, and the gestational stage at which pregnancy loss occurred. These results, which confirm unusual features of BTV-8 infection, should not be extrapolated to infection with other BTV strains without thorough evaluation. This study also adds weight to the hypothesis that the re-emergence of BTV-8 in France in 2015 may be attributable to the use of contaminated bovine semen.

JAK3 and PI3K mediates the suppressive effects of interferon tau on neutrophil extracellular traps formation during peri-implantation period

Interferon tau (IFNτ) is the main maternal signal for pregnancy in ruminants and modulates the functions of various immune cells, including neutrophils. Neutrophil extracellular traps (NETs) are one of the main defence mechanisms of neutrophils. In this study, we observed higher (p < 0.01) ex-vivo NETs extrusion by blood neutrophils from day 16–18 post artificial insemination (AI) in non-inseminated and inseminated non-pregnant cows compared to pregnant cows. In vitro study also showed that IFNτ hampers NETs formation in dose and time dependent manner. The lowest (p < 0.01) NETs formation and the highest (p < 0.01) mRNA expression (RT-PCR) of IFNτ stimulated genes (ISG15, OAS1, MX1) were observed when neutrophil incubated with 9 ng/mL IFNτ for 3.5 h. Signalling cascades mediating IFNτ impairment of NETs formation were identified using inhibitors of JAK2, JAK3, p38, PI3K/Akt and MAPK/Erk. IFNτ reduced (p < 0.01) the mRNA expression (RT-PCR) and concentration (ELISA) of genes and proteins that mediate NETs formation in blood neutrophils including histones (H1, H2), neutrophil elastase (NE) and myeloperoxidase (MPO). However, the effects of IFNτ on these genes and proteins were eliminated in the presence of JAK3 or PI3K inhibitors. Immunocytochemistry study also showed strong MPO signal in the presence of JAK3 or PI3K inhibitors as compared to positive control (PC, IFNτ alone). The results indicate that IFNτ impairs NETs formation using JAK3 and PI3K and thus essential for successful implantation and establishment of pregnancy in cows.

Early embryo losses, progesterone and pregnancy associated glycoproteins levels during summer heat stress in dairy cows.

Objectives of this study were to characterize the effects of heat stress on pregnancy associated glycoproteins (PAG) and progesterone and its involvement in embryo survival. In trial 1, blood samples collected from days 29 to 36 post insemination were examined for the comparison of PAG concentrations between winter (n = 3721) and summer (n = 2388). In trial 2, embryo losses were assessed in winter (n = 144) and in summer (n = 133), in days 31 or 32 of pregnancy. Pregnancy diagnosis was carried out by ultrasonography on days 24 or 25, and it was repeated a week later; in the second occasion PAG concentration was also determined. In trial 3 the PAG and progesterone concentrations were assessed in days 33 to 36 in winter and summer. In trial 1 PAG levels did not differ between winter and summer, the conception rate and the proportion of uncertain pregnancies were higher in winter than summer. The likelihood of pregnancy was 10 to 15% higher in winter. In trial 2, the embryo death rate was higher in summer, but the PAG levels of cows that had embryo loss in summer were higher than those in winter. In both seasons, lower PAG levels were associated with higher risk of pregnancy loss, while embryo death was five times more likely to occur in summer than in winter and lower PAG concentrations were positively associated with higher risk of embryo loss. In trial 3, mean PAG levels were higher and of progesterone were lower during the summer than during the winter. We infer that despite the devastating effects of heat stress on cows' fertility, those early embryos that survive under continuous heat stress can form a well-functioning placenta; hence, the high embryo mortality rate observed during the summer months could be mainly attributed to luteal insufficiency.

Curtailing the conventional dose of Folltropin-V for superstimulation and embryo recovery in Sahiwal cattle (Bos indicus)

High cost of the follicle stimulating hormone (FSH) impacted embryo transfer technology (ETT) work in India. The experiment evaluated effects of reduced (200 mg vs 400 mg) FSH (Folltropin-V) dose on superovulation and embryo recovery in Sahiwal cattle. Animals treated with conventional dose of Folltropin, i.e. 400 mg (G1, n=6) were compared with another group administered with half dose (i.e. 200 mg) of Folltropin (G2, n=6). Superstimulatory response (number of follicles &gt;8 mm) during estrus; unovulatory follicles, superovulatory response (one day prior to embryo flushing) using trans-rectal ultrasonography were recorded. Embryos were collected nonsurgically on day-7 post insemination and total recovered and transferable embryos were noted. Results revealed that number of follicles during estrus and subsequent ovulations were significantly higher in the G1 compared to G2 (30.0±1.37 and 26.3±0.99 vs. 20.3±0.21 and 17.7±0.42, respectively). Unovulatory follicles per animal were lower (2.6±0.21 vs. 3.7±0.49) in G2 than G1. The group receiving lower dose of Folltropin (G2) had significantly higher average embryo recovery compared to G1 (12.0±0.63 vs 8.7±0.76, respectively). Additionally, average number of transferable embryos were non-significantly higher in G2 (7.0±0.37) than G1 (5.7±0.88). The study indicated that lower dose of Folltropin-V (200 mg compared to 400 mg) is more effective in superovulation and embryo production in Sahiwal cows.

Effect of various hCG treatment protocols on luteal characteristics, plasma progesterone concentration, and pregnancy in normal cyclic Indian crossbred dairy cows.

Limited information exists on the application of human chorionic gonadotropin (hCG) post insemination in the Indian crossbred dairy cows. This study aimed to evaluate the effect of four hCG administration protocols on luteal characteristics and pregnancy outcomes following artificial insemination (AI). Using block randomization, 50 healthy lactating crossbred cows were allocated in equal numbers (n = 10 cows per group) to receive either 5 mL normal saline (control) or 1500 IU hCG on the day of AI (hGG-0), day 7 post AI (hCG-7), day 14 post AI (hCG-14), or days 0, 7, and 14 post AI (hCG-0,7,14). All cows were scanned using sequential transrectal ultrasound examinations to evaluate primary luteal parameters, development of accessory corpora lutea, and pregnancy. Serial blood samples were collected to measure plasma progesterone concentrations. Data were analyzed using repeated measures ANOVA and Fisher's exact tests. The mean primary luteal area, total luteal area, and total luteal diameter values were significantly greater in the hCG-treated cows. Compared to the control, the hCG-14 group had a significantly higher percentage of cows with an accessory corpora luteum. However, there were no significant differences in the mean progesterone concentrations or the first service conception rates between any of the groups. Overall, the results of this study indicate that while hCG administration post AI in healthy Indian crossbred cows may enhance primary luteal dimensions or induce the formation of accessory corpora lutea, it does not appear to have any beneficial effect on luteal function or pregnancy.

Analysis of Morphokinetic Parameters of Feline Embryos Using a Time-Lapse System.

Simple SummaryThis study was conducted with the aim of analyzing the morphokinetic parameters that determine the proper development of feline embryos in vitro. Our research was carried out using a time-lapse monitoring system shows that the timing of the first and second cleavage divisions, the timing of blastocyst cavity formation and morphological anomalies can all be used as early and non-invasive indicators of cat embryo development in vitro.The aim of this study was to analyze the morphokinetic parameters of feline embryos using a time lapse system. Oocytes matured in vitro were fertilized (IVF) and in vitro cultured in a time lapse-system (Primo Vision®, Gothenburg, Sweden). The first cell division of embryos occurred between 17 h post insemination (hpi) and 38 hpi, with the highest proportion of embryos (46%) cleaving between 21 and 24 hpi. The timing of the first cleavage significantly affected further embryo development, with the highest development occurring in embryos that cleaved at 21–22 hpi. Embryos that cleaved very early (17–18 hpi) developed poorly to the blastocyst stage (2%) and none of the embryos that cleaved later than 27 hpi were able to reach the blastocyst stage. Morphological defects were observed in 48% of the embryos. There were no statistically significant differences between the timing intervals of the first cleavage division and the frequency of morphological defects in embryos. Multiple (MUL) morphological defects were detected in more than half (56%) of the abnormal embryos. The most frequent single morphological defects were cytoplasmic fragmentation (FR) (8%) and blastomere asymmetry (AS) (6%). Direct cleavage (DC) from 1–3 or 3–5 blastomeres, reverse cleavage (RC) and vacuoles were rarely observed (2–3%). The timing of blastocyst cavity formation is a very good indicator of embryo quality. In our study, blastocyst cavity formation occurred between 127–167 hpi, with the highest frequency of hatching observed in blastocysts that cavitated between 142–150 hpi. Blastocysts in which cavitation began after 161 h did not hatch. In conclusion, the timing of the first and second cleavage divisions, the timing of blastocyst cavity formation and morphological anomalies can all be used as early and non-invasive indicators of cat embryo development in vitro.