Transfer Of In Vitro-produced Embryos Research Articles

The impact of in vitro embryo production on placental and umbilical cord vascularization is minimized by the addition of reproductive fluids

Animals born from in-vitro-produced (IVP) embryos show changes in the placenta and umbilical cord vascularization. This study compares the placental and umbilical vascular morphometry in pigs (n = 19) born through artificial insemination (AI group) or after transfer of IVP embryos cultured with (RF-IVP group) or without (C-IVP group) reproductive fluids. The relationship between vascular parameters and animal growth during the first year of life was also analyzed. Samples were collected at birth, fixed, paraffin-embedded, cut in sections, stained, and photographed for vascular and morphometric analysis with ImageJ® and Slide Viewer®. The average daily weight gain was individually scored from birth to the first year of life. No differences were found in placental vascular morphometry among groups, except for the vascular area of small vessels (arterioles, venules, and small vessels) that was higher in the C-IVP group. Regarding the umbilical cord, the values for perimeter (AI: 26.40 ± 3.93 mm; IVP: 30.51 ± 4.74 mm), diameter (AI: 8.35 ± 1.01 mm; IVP: 10.26 ± 1.85 mm), area (AI: 43.18 ± 12.87; IVP: 56.61 ± 14.89 mm2), and Wharton's jelly area (AI: 36.86 ± 12.04 mm2; IVP 48.88 ± 12.80 mm2) were higher in IVP-derived than AI-derived animals, whereas arterial and venous morphometric data were similar between groups. A correlation study showed that placental and umbilical cord vascular phenotypes affect the further growth of pigs. In conclusion, assisted reproductive technologies impact small caliber vessels in the placenta and morphometric parameters in the umbilical cord. The addition of reproductive fluids in IVP-embryo contributes to reduce the differences with in vivo-derived animals.

Economic analysis of the use of in vitro produced embryos transferred during heat stress under dairy herd constraints

The use of embryo transfer helps to improve reproductive performance during periods of heat stress. In vitro produced embryo transfer (IVP-ET) is more expensive than artificial insemination. We hypothesized that the value IVP-ET in seasonal herds depends on herd constraints, such as the maximum number of milking cows and the maximum number of calvings that can be accommodated throughout the year. Therefore, the objective of this study was to estimate how profitability in dairy herds exposed to summer heat stress is affected by the number of months in which IVP-ET is used, the use of IVP-ET in repeat-breeder cows, IVP-ET cost, and herd constraints. We built and used a nonlinear programming model of a dairy herd with young stock and cows with monthly Markov Chain transitions. The model varied the number of heifers calving in each calendar month to maximize herd profitability. We varied IVP-ET cost ($100 or $200), duration of the IVP-ET program (2 or 4 months), and the breeding number in which IVP-ET started (1st or 3rd). In total, 20 scenarios were simulated. Maximum profitability was obtained when IVP-ET was not used, regardless of herd constraints. The 16 scenarios in which IVP-ET was used showed increased seasonality in milk yield, numbers of milking cows, total cows, total calvings, and heifer calvings because the program tried to limit the number of IVP-ET breedings in the summer. The addition of the calving constraint increased the value of IVP-ET. The breakeven cost per IVP-ET ranged from −$6.79 to $24.38 compared with conventional semen cost of $20. In conclusion, the current market costs of IVP-ET did not warrant application with the objective to increase reproductive performance during heat stress. Herd constraints on the maximum allowable seasonality in the monthly number of milking cows and calvings affected the value of IVP-ET during heat stress.

Characterization of the endometrial transcriptome in early diestrus influencing pregnancy status in dairy cattle after transfer of in vitro-produced embryos.

Modifications of the endometrial transcriptome at day 7 of the estrus cycle are crucial to maintain gestation after transfer of in vitro-produced (IVP) embryos, although these changes are still largely unknown. The aim of this study was to identify genes, and their related biological mechanisms, important for pregnancy establishment based on the endometrial transcriptome of recipient lactating dairy cows that become pregnant in the subsequent estrus cycle, upon transfer of IVP embryos. Endometrial biopsies were taken from Holstein Friesian cows on day 6-8 of the estrus cycle followed by embryo transfer in the following cycle. Animals were classified retrospectively as pregnant (PR, n = 8) or nonpregnant (non-PR, n = 11) cows, according to pregnancy status at 26-47 days. Extracted mRNAs from endometrial samples were sequenced with an Illumina platform to determine differentially expressed genes (DEG) between the endometrial transcriptome from PR and non-PR cows. There were 111 DEG (false discovery rate < 0.05), which were mainly related to extracellular matrix interaction, histotroph metabolic composition, prostaglandin synthesis, transforming growth factor-β signaling as well as inflammation and leukocyte activation. Comparison of these DEG with DEG identified in two public external data sets confirmed the more fertile endometrial molecular profile of PR cows. In conclusion, this study provides insights into the key early endometrial mechanisms for pregnancy establishment, after IVP embryo transfer in dairy cows.

Piglets produced by transfer of embryos obtained by in vitro fertilization of oocytes matured in vitro with thymosin: A case report

The aim of the study was to examine the in vivo viability of in vitro-produced (IVP) porcine embryos obtained from oocytes matured with thymosin. The research material for this study consisted of immature pig oocytes obtained from ovaries after slaughter and ejaculated semen obtained from one boar. The immature oocytes were cultured in vitro until the metaphase II stage in a medium supplemented with thymosin (TMS). The presumptive zygotes obtained were cultured in vitro for 4-40 hours. The presumptive zygotes and 2-4-cell embryos were evaluated in vivo after transferring them to synchronized recipients. After the transfer of embryos from the experimental group into 2 recipients (50 embryos into each gilt) and the transfer of 50 embryos from the control group into 1 recipient, both gilts that had received embryos obtained by in vitro fertilization of oocytes matured with TMS became pregnant and delivered a total of 16 live piglets. After the transfer of embryos from the control group, no pregnancy was achieved. In conclusion, the results of our preliminary study suggest that the maturation of pig oocytes with thymosin supports the in vivo survival of in vitro produced embryos. It is important to note, that this was the first birth of piglets obtained after transfer of IVP embryos in Poland.

8 Relationship between oestrus manifestation and pregnancy rates on recipients used in an invitro-produced embryo transfer program

The main objective of an embryo transfer (ET) program is to increase the genetic value of the offspring produced in a given herd. Nutrition, management, and efficiency in the detection of oestrus are among the factors that affect the use of this technology. Protocols that induce oestrus or ovulation allow practitioners to transfer embryos in a predetermined time without oestrus detection (Bó et al. 2012 Reprod. Fertil. Dev. 24, 272-277; https://doi.org/10.1071/RD11918). Although these protocols have been performed adequately for several years, recent attention has been directed to the effect of oestrus expression and circulating oestradiol concentrations during the growth of the preovulatory follicle on embryo development and pregnancy. Oestrus expression is positively associated with high pregnancies per ET (P/ET) and reduced pregnancy losses in recipients receiving invitro-produced (IVP) bovine embryos (Bó and Cedeño 2018 Anim. Reprod. 15, 224-230; https://doi.org/10.21451/1984-3143-AR2018-0060). The present study was designed to evaluate the relationship between time of oestrus manifestation and P/ET on recipients involved in an IVP ET program. A total of 189 multiparous Aberdeen Angus and Girolando cows were used as recipients. The protocol consisted of an intravaginal progesterone (P4) implant (Eazi-Breed CIDR, Zoetis) and gonadorelin (100μg) treatment on Day 0 and dinoprost (25mg) treatment, placement of an Estrotect patch to improve oestrus observation, and CIDR removal on Day 7. Oestrus observation occurred every 12h from 36 to 96h after CIDR removal, and ET was performed on Day 17 and 18. Fresh IVP embryos were transferred after being cultured in standard synthetic oviductal fluid with amino acids with 3% bovine serum (Rubessa et al. 2011 Theriogenology 76, 1347-1355; https://doi.org/10.1016/j.theriogenology.2011.06.004). Only quality 1 and 2 embryos (IETS) were transferred. Embryo stages 4, 5, 6, and 7 (IETS) were randomly assigned to the recipients. No comparison between embryo grade and oestrus observation was evaluated. The cows were sorted according to oestrus observation in the following groups: 36, 48, 60, 72, 84, and 96h after CIDR removal. Pregnancy diagnosis was conducted 25 days after ET by ultrasonography. A chi-squared test with Fisher correction was used to evaluate the data set. Differences were considered significant if P&amp;lt;0.05. Out of the 189 cows submitted to the synchronization protocol, 160 were detected in oestrus (84.66%), 17 (8.99%) did not show oestrus, and 12 (6.35%) lost their Estrotect patches. Considering all recipients (with or without oestrus expression), oestrus manifestation was greater within the 48- to 60-h interval (40/189; 21.16% for both). The overall P/ET was 41.27% (78/189), and there was no statistical difference among groups (time of oestrus after P4 implant removal) for P/ET. Pregnancies per ET of the recipients that did not show oestrus was 35.29% (6/17), and P/ET for the ones that lost their Estrotect was 33.33% (4/12). Therefore, we concluded that all recipients showing oestrus within an interval from 36 to 96h after P4 implant removal may be used in an ET program using IVF embryos without affecting overall P/ET.

100 Relationship between corpus luteum blood flow evaluated via colour Doppler ultrasound and pregnancy rate in bovine embryo transfer recipients

The advent of colour Doppler ultrasound has allowed evaluation of blood flow to bodily tissues. This novel technology is being tested as a tool to improve efficiency in a variety of livestock reproduction programs. The objective of the present study was to evaluate the relationship between blood flow to the ovarian corpus luteum (CL), imaged via colour Doppler ultrasound, and pregnancy rate in crossbred dairy recipients for embryo transfer (ET). Oestrous cycles of Bos indicus×Bos taurus dairy heifers (n=90, 16-24 months of age) were synchronized for embryo transfer on Days 7-8. Immediately before ET, heifers were palpated for the presence of ovarian CL and CL papillae. Presence of CL, CL papillae, and CL lacunae were confirmed via transrectal B-mode ultrasound (Ibex EVO I, E. I. Medical Imaging). Transrectal colour Doppler ultrasound (Ibex EVO I, E. I. Medical Imaging) was used to evaluate blood flow to the CL. Invitro-produced (IVP) blastocysts (qualities I and II) were transferred to the uterine horn ipsilateral to the CL. All palpation, imaging, and embryo transfer was performed by a single technician. Pregnancy was determined via transrectal ultrasound 24-32 days post-embryo transfer (31-39 days of embryo age). Blood flow to the CL was split into three categories: high, median, and low. Presence or absence of two additional CL structures, CL papillae and lacunae, were recorded. Fisher's exact test was used to evaluate data. Statistical differences were considered significant at P&amp;lt;0.05. The overall pregnancy rate was determined to be 40%, 36 out of 90. Pregnancy rates were similar in high (33.9%, 20 of 59), median (59.09%, 13 of 22), and low (33.33%, 3 of 9) blood flow categories. Consistent with previous studies, absence of CL papillae and presence of CL lacunae at the time of ET did not affect pregnancy rate. Doppler blood flow, papillae, and lacunae were also evaluated together in each possible combination. Only one outlier was present; all other differences were not significant. The highest pregnancy rate (66.67%, 10 of 15) was observed in recipients with CLs with median Doppler blood flow, a palpable CL papillae, and no CL lacunae. A larger sample size is required to determine the accuracy of this measurement. In conclusion, recipients with high to low CL blood flow are suitable for IVP embryo transfer programs. Alone, colour Doppler imaging of the CL does not predict pregnancy rate.

Zinc supplementation during in vitro embryo culture increases inner cell mass and total cell numbers in bovine blastocysts1.

Deficiencies in current embryo culture media likely contribute to the poor blastocyst development rates and pregnancy retention rates for in vitro produced (IVP) bovine embryos. Of special concern is the lack of micronutrients in these media formulations. One micronutrient of interest is zinc, an essential trace element involved with various enzyme and transcription factor activities. The objective of this work was to describe whether zinc sulfate supplementation during in vitro embryo culture affects bovine embryo development and blastomere numbers. Either 0, 2, 20, or 40 µM zinc sulfate was supplemented to presumptive zygotes cultured in synthetic oviductal fluid containing AAs and bovine serum albumin for 8 d. None of the treatments affected cleavage rates. Percentage of blastocysts on days 7 and 8 postfertilization was not affected by supplementing 2 or 20 µM zinc but were reduced (P < 0.05) with 40 µM zinc. In blastocysts harvested on day 8, inner cell mass (ICM) and total cell number were increased (P < 0.05) with 2 µM zinc supplementation but not with the other zinc concentrations. Numbers of trophectoderm cells were not affected by zinc treatment. In conclusion, supplementing zinc during bovine embryo culture did not impact blastocyst development but improved ICM cell numbers. This improvement in ICM cell number may have implications for improved pregnancy retention rates after IVP embryo transfer as smaller ICM sizes are associated with poor pregnancy success in cattle.

In vitro production of horse embryos predisposes to micronucleus formation, whereas time to blastocyst formation affects likelihood of pregnancy.

Invitro embryo production is an increasingly popular means of breeding horses. However, success is limited by a high incidence of early embryo loss. Although there are various possible causes of pregnancy failure, chromosomal abnormalities, including aneuploidy, are important potential contributors. This study evaluated the frequency of micronucleus formation as a proxy for aneuploidy in invitro-produced (IVP) and invivo-derived horse blastocysts. Associations between IVP embryo morphology, frequency of nuclear abnormalities and the likelihood of pregnancy were investigated. IVP blastocysts exhibited a higher frequency of cells with micronuclei than invivo-derived embryos (10% vs 1% respectively; P=0.05). This indication of chromosomal instability may explain the higher incidence of pregnancy failure after transfer of IVP embryos. However, the frequency of micronuclei was not correlated with brightfield microscopic morphological characteristics. Nevertheless, IVP embryos reaching the blastocyst stage after Day 9 of invitro culture were less likely to yield a pregnancy than embryos that developed to blastocysts before Day 9 (27% vs 69%), and embryos that had expanded before transfer were more likely to undergo embryonic death than those that had not expanded (44% vs 10%). These findings indicate that current embryo culture conditions are suboptimal and that the speed of embryo development is correlated with pregnancy survival.

Update on the vitrification of bovine oocytes and invitro-produced embryos.

The combined use of reproductive technologies, such as transvaginal ovum-pick up and invitro embryo production followed by direct transfer of cryopreserved embryos, has great potential for enhancing genetic selection and optimising cross-breeding schemes in beef and dairy cattle production systems. This, along with an effective cryopreservation procedure for cow oocytes, will enable the long-term conservation of female genetic traits and the advance of embryo biotechnology in this species. However, the low fertilisation rates and developmental competence of cryopreserved oocytes still need to be improved. Over the past two decades, many research efforts tried to overcome individual features of the bovine oocyte that make it notoriously difficult to cryopreserve. In addition, pregnancy rates associated with invitro-produced (IVP) embryos remain lower than those obtained using invivo counterparts. This, together with a lack of a standard methodology for IVP embryo cryopreservation that provides easier and more practical logistics for the transfer of IVP embryos on farms, has hindered international genetic trade and the management of embryo banks. This review updates developments in oocyte and IVP embryo vitrification strategies targeting high production efficiency and better outcomes.

81 Effects of Day 5 or 6 HEPES-Buffered Transportation Culture Medium on Developmental Competence of Bovine In Vitro-Produced Embryos

Most in vitro-produced (IVP) bovine embryos are transferred fresh. Use of a HEPES/bicarbonate embryo culture medium for transportation would offer flexibility for embryo shipment and transfer. We hypothesized that embryos cultured for 36 (Day 6 embryos) or 60 h (Day 5 embryos) in a novel SCF1T medium (SOF for Conventional Freezing 1 supplemented with HEPES) would maintain developmental competence compared with bicarbonate-buffered medium SCF1 (control). In 5 replicates, IVP embryos were produced by aspirating cumulus–oocyte complexes (COC) from 2-to 8-mm follicles of abattoir ovaries. The COC (n = 1036) were matured for 23 h, fertilized with semen from 1 of 3 bulls, and cultured in SCF1 at 38.5°C, 5% CO2, 5% O2, and 90% N2 (Owen et al. 2017 Reprod. Fertil. Dev. 29, 129-130). Randomly, on Day 5 and 6 after fertilization, a subset of presumptive embryos were moved into 500-µL polystyrene vials containing 100 µL of SCF1T medium, covered with 300 µL of sterile mineral oil and cultured in a portable incubator (MicroQ iQ2, Scottsdale, AZ, USA) at 38.5°C for 60 and 36 h, respectively. On Day 7.5 post-fertilization, blastocyst rates were evaluated and embryos (n = 8) from each group were stained with 1 µg mL−1 Nile Red for lipid quantification, and 300 nM Mitotracker Red CMX-Rosamine (Thermo Fisher Scientific, Waltham, MA, USA) for mitochondrial polarity. Images were obtained with confocal microscopy and fluorescent intensity (AFU) was measured by Image J software (National Institutes of Health, Bethesda, MD, USA). Data were analysed by one-way ANOVA and means separated by Tukey’s HSD. Results (Table 1) indicate similar blastocyst rates and lipid content between embryos cultured for 36 to 60 h in SCF1T and control media (P &gt; 0.05). However, mitochondrial polarity was lower in the Day 5 group (P &lt; 0.05) compared with Day 6 and control groups. Results suggest that culturing embryos in SCF1T medium for 36 h maintains developmental competence compared with bicarbonate-buffered media and offers an alternative for shipment and transfer of IVP embryos. Studies involving evaluation of pregnancy rates of the present study are ongoing. Table 1.Effects of Day 5 or 6 SCF1T embryo culture medium on development, lipid content, and mitochondrial polarity

Economic and genetic performance of various combinations of in vitro-produced embryo transfers and artificial insemination in a dairy herd

The objective of this study was to find the optimal proportions of pregnancies from an in vitro-produced embryo transfer (IVP-ET) system and artificial insemination (AI) so that profitability is maximized over a range of prices for embryos and surplus dairy heifer calves. An existing stochastic, dynamic dairy model with genetic merits of 12 traits was adapted for scenarios where 0 to 100% of the eligible females in the herd were impregnated, in increments of 10%, using IVP-ET (ET0 to ET100, 11 scenarios). Oocytes were collected from the top donors selected for the trait lifetime net merit (NM$) and fertilized with sexed semen to produce IVP embryos. Due to their greater conception rates, first ranked were eligible heifer recipients based on lowest number of unsuccessful inseminations or embryo transfers, and then on age. Next, eligible cow recipients were ranked based on the greatest average estimated breeding values (EBV) of the traits cow conception rate and daughter pregnancy rate. Animals that were not recipients of IVP embryos received conventional semen through AI, except that the top 50% of heifers ranked for EBV of NM$ were inseminated with sexed semen for the first 2 AI. The economically optimal proportions of IVP-ET were determined using sensitivity analysis performed for 24 price sets involving 6 different selling prices of surplus dairy heifer calves at approximately 105 d of age and 4 different prices of IVP embryos. The model was run for 15 yr after the start of the IVP-ET program for each scenario. The mean ± standard error of true breeding values of NM$ of all cows in the herd in yr 15 was greater by $603 ± 2 per cow per year for ET100 when compared with ET0. The optimal proportion of IVP-ET ranged from ET100 (for surplus dairy heifer calves sold for ≥$300 along with an additional premium based on their EBV of NM$ and a ≤$100 embryo price) to as low as ET0 (surplus dairy heifer calves sold at $300 with a $200 embryo price). For the default assumptions, the profit/cow in yr 15 was greater by $337, $215, $116, and $69 compared with ET0 when embryo prices were $50, $100, $150, and $200. The optimal use of IVP-ET was 100, 100, 62, and 36% of all breedings for these embryo prices, respectively. At the input price of $165 for an IVP embryo, the difference in the net present value of yr 15 profit between ET40 (optimal scenario) and ET0 was $33 per cow. In conclusion, some use of IVP-ET was profitable for a wide range of IVP-ET prices and values of surplus dairy heifer calves.

Comparison between an exclusive in vitro–produced embryo transfer system and artificial insemination for genetic, technical, and financial herd performance

The objective of this study was to implement an in vitro-produced embryo transfer (IVP-ET) system in an existing stochastic dynamic dairy simulation model with multitrait genetics to evaluate the genetic, technical, and financial performance of a dairy herd implementing an exclusive IVP-ET or artificial insemination (AI) system. In the AI system, sexed semen was used on the genetically best heifers only. In the IVP-ET system, all of the animals in the herd were impregnated with female sexed embryos created through in vitro fertilization of oocytes collected from animals of superior genetics for different traits of interest. Each donor was assumed to yield on average 4.25 transferable embryos per collection. The remaining animals in the herd were used as recipients and received either a fresh embryo or a frozen embryo when fresh embryos were not available. Selection of donors was random or based on the greatest estimated breeding value (EBV) of lifetime net merit (NM$), milk yield, or daughter pregnancy rate. For both the IVP-ET and AI systems, culling of surplus heifer calves not needed to replace culled cows was based on the lowest EBV for the same traits. A herd of 1,000 milking cows was simulated 15 yr over time after the start of the IVP-ET system. The default cost to produce and transfer 1 embryo was set at $165. Prices of fresh embryos at which an exclusive IVP-ET system financially breaks even with the comparable AI system in yr 15 and for an investment period of 15 yr were also estimated. More surplus heifer calves were sold from the IVP-ET systems than from the comparable AI systems. The surplus calves from the IVP-ET systems were also genetically superior to the surplus calves from the comparable AI systems, which might be reflected in their market value as a premium price. The most profitable scenario among the 4 IVP-ET scenarios in yr 15 was the one in which NM$ was maximized in the herd. This scenario had an additional profit of $8/cow compared with a similar AI scenario that maximized NM$, provided that surplus heifer calves could be sold at a premium price based on the superiority of the EBV of NM$. For the IVP-ET system to be at least as profitable as the comparable AI system during a 15-yr investment period, the surplus calves from the IVP-ET system needed to be sold at the premium prices. The break-even price of fresh embryos was estimated to be $84 for the exclusive IVP-ET system. This resulted in the same profit as the AI system, which maximized NM$ for a 15-yr investment period and in which heifer calves were sold at a premium price.

Evidence of Placental Autophagy during Early Pregnancy after Transfer of In Vitro Produced (IVP) Sheep Embryos.

Pregnancies obtained by Assisted Reproductive Technologies (ART) are associated with limited maternal nutrient uptake. Our previous studies shown that in vitro culture of sheep embryos is associated with vascularization defects in their placentae and consequent reduction of embryo growth. Autophagy is a pro-survival cellular mechanism triggered by nutrient insufficiency. Therefore, the goal of our present study was to determine if autophagy is involved in early placental development after transfer of in vitro produced (IVP) embryos. To do this, placentae obtained following transfer of IVP sheep embryos were compared with placentae obtained after natural mating (control—CTR). The placentae were collected on day 20 post-fertilization and post-mating, respectively, and were analyzed using molecular (qPCR), ultrastructural and histological/immunological approaches. Our results show drastically increased autophagy in IVP placentae: high levels of expression (p<0.05) of canonical markers of cellular autophagy and a high proportion of autophagic cells (35.08%; p<0.001) were observed. We conclude that high autophagic activity in IVP placentae can be a successful temporary counterbalance to the retarded vasculogenesis and the reduction of foetal growth observed in pregnancies after transfer of IVP embryos.

A new direct transfer protocol for cryopreserved IVF embryos

The global demand for in vitro–produced (IVP) embryos of determined sex has greatly increased over the last decade. Efficient protocols for the direct transfer of IVP embryos are lacking. This study aimed to compare the pregnancy rates for fresh, vitrified, or frozen/directly transferred IVP dairy cow embryos. Oocytes (n = 3171) recovered by ovum pickup (n = 112) from Girolando (Holstein–Gir) females (n = 36) were selected and submitted to IVM for 24 hours at 38.5 °C with 5% CO2 in air with saturated humidity. In vitro fertilization was performed with the thawed, sexed semen from 5 Holstein bulls. After IVF, presumptive zygotes were denuded and cultured for 7 days under the same IVM and IVF conditions of temperature and humidity, except with 5% CO2 and 5% O2. Grade I blastocysts were randomly assigned for either the transferred fresh, vitrified/thawing, or frozen/directly embryo transfer into previously synchronized recipient females. Conception rates were analyzed by binomial logistic regression, and a probability level of P < 0.05 was considered significant. The conception rates were 51.35 ± 1.87% (133/259) for the fresh embryos, 35.89 ± 3.87% (84/234) for the vitrified embryos, and 40.19 ± 4.65% (125/311) for the frozen directly transferred embryos. These data demonstrate that IVP embryos with sexed semen could be directly transferred into recipient cows with similar conception rates to vitrified embryos. The comparison found that the use of frozen embryos in direct transfer provides easier logistics and a more practical approach for the transfer of IVP embryos on dairy farms.

Pregnancy Rates Bovine Recipients Inovulated with in Vitro Produced (IVP) Embryos in The Legal Amazon

The aim of the work was to evaluate transfer efficiency in in vitro produced (IVP) bovine embryos, at different stages of development, through pregnancy rates of inovulated cows, in the Amazon Region. 5403 embryo transfers conducted during dry and rain season, with IVP embryos, at different stages of development, and recipients with different corpora lutea types, were evaluated. The oocytes were obtained from zebu cows, through vaginal aspiration by ultrasonography. The oocytes were matured, fertilized, and cultured in vitro. The embryos were transferred to cyclic crossbred cows, at the stages of morula (MO), early blastocyst (BI), blastocyst (BL), expanded blastocyst (BX), hatching blastocyst (BN) and hatched blastocyst (EB). The corpora lutea were classified by ultrasonography into protruded and included. Pregnancy was achieved in 34.30% (1855) of the animals; with significantly higher pregnancy rates for embryos at BX (38.2%) stage. The embryos at BN and BX stages were similar; however MO, BI and BL stages did not differ statistically (P>0.05). The analysis showed no correlation between corpus luteum size, or type, and the development of IVP embryos inovulated at different stages. Pregnancy rates did not differ (P > 0.05) for embryo transfers performed during dry (33.7%) or rainy (35.7%) season. Pregnancy outcomes obtained after transfer of IVP embryos, from the Amazon Region, were similar to studies in other regions and were not influenced by seasons.

Embryonic and calving losses in bovine mixed-breed twins induced by transfer of in vitro-produced embryos to bred recipients

One or two in vitro-produced (IVP) Japanese Black (JB) cattle embryos at 8 days after in vitro fertilization were transferred to the contralateral uterine horn of previously bred Japanese Shorthorn (JSH) or JSH–JB cross recipients, and then the occurrence of early embryonic death, abortion during mid- and late gestation, and calving loss were recorded. The survival rate of embryos, including indigenous ones, was not affected by the number of embryos transferred, and a significantly higher twinning rate (68% of pregnant recipients at 80 days after transfer) was achieved when two IVP embryos were transferred, as compared with the rate when one IVP embryo was transferred (24%). In late ET (recipients at 8.5–9.0 days after the onset of oestrus), the embryo survival rate (22%) and the pregnancy rate (42%) at 80 days after ET were significantly lower than those rates in the synchronous ET (recipients at 8.0 days after the onset of oestrus; 47 and 79%, respectively). In the early ET (recipients at 6.0–7.5 days after the onset of oestrus), no significant differences from the synchronous ET were detected in these rates. Twenty-six percent of twin pregnant recipients were aborted during mid- or late-pregnancy, and 39% of twin calves were stillborn. The mean gestation length of the twin-bearing JSH dams (276 days) was 1 week shorter than that of the single-bearing JSH dams, and it was 2 weeks shorter than that of the JB dams bearing a single JB calf derived from the IVP embryos. The longer gestation length of single JB calves derived from IVP embryos resulted in a significantly higher mean birth weight than that of in vivo control calves with the standard length of gestation. In conclusion, the number of embryos to be transferred did not affect the embryo survival rate, and the transfer of two IVP embryos to previously inseminated recipients induced a significantly higher twinning rate during early pregnancy than that of one IVP embryo transfer. The incidence of embryonic losses during early pregnancy increased when Day 8 embryos were transferred to the recipients later in the oestrous cycle (>8.0 days). The results suggested that one cause of the high rate of abortions and stillbirths in twin-bearing dams is the difference in the mean gestation length between the native JSH and JB foetuses derived from transferred IVP embryos.

Numbers of small follicles in ovaries affect superovulation response in cattle

The global demand for in vitro–produced (IVP) embryos of determined sex has greatly increased over the last decade. Efficient protocols for the direct transfer of IVP embryos are lacking. This study aimed to compare the pregnancy rates for fresh, vitrified, or frozen/directly transferred IVP dairy cow embryos. Oocytes (n = 3171) recovered by ovum pickup (n = 112) from Girolando (Holstein–Gir) females (n = 36) were selected and submitted to IVM for 24 hours at 38.5 °C with 5% CO2 in air with saturated humidity. In vitro fertilization was performed with the thawed, sexed semen from 5 Holstein bulls. After IVF, presumptive zygotes were denuded and cultured for 7 days under the same IVM and IVF conditions of temperature and humidity, except with 5% CO2 and 5% O2. Grade I blastocysts were randomly assigned for either the transferred fresh, vitrified/thawing, or frozen/directly embryo transfer into previously synchronized recipient females. Conception rates were analyzed by binomial logistic regression, and a probability level of P < 0.05 was considered significant. The conception rates were 51.35 ± 1.87% (133/259) for the fresh embryos, 35.89 ± 3.87% (84/234) for the vitrified embryos, and 40.19 ± 4.65% (125/311) for the frozen directly transferred embryos. These data demonstrate that IVP embryos with sexed semen could be directly transferred into recipient cows with similar conception rates to vitrified embryos. The comparison found that the use of frozen embryos in direct transfer provides easier logistics and a more practical approach for the transfer of IVP embryos on dairy farms.