In Vitro Embryo Production Research Articles

Bovine in vitro oocyte maturation and embryo culture in liquid marbles 3D culture system.

Despite the advances in in vitro embryo production (IVP) over the years, the technique still has limitations that need to be overcome. In cell cultures, it is already well established that three-dimensional culture techniques are more physiological and similar to the in vivo development. Liquid marble (LM) is a three-dimensional system based on the use of a hydrophobic substance to create in vitro microbioreactors. Thus, we hypothesized that the LM system improves bovine in vitro oocyte maturation and embryo culture. In experiment I, bovine cumulus-oocyte complexes (COCs) were placed for in vitro maturation for 22h in two different groups: control (conventional 2D culture) and LM (three-dimensional culture). We found that oocyte nuclear maturation was not altered by the LM system, however it was observed a decrease in expression of genes important in the oocyte maturation process in cumulus cells of LM group (BCL2, EIF4E, and GAPDH). In experiment II, the COCs were conventionally matured and fertilized, and for culture, they were divided into LM or control groups. There was a decrease in blastocyst rate and cell counting, a down-regulation of miR-615 expression, and an increase in the DNA global methylation and hydroxymethylation in embryos of LM group. Therefore, for the bovine in vitro embryo production, this specific three-dimensional system did not present the advantages that we expected, but demonstrated that the embryos changed their development and epigenetics according to the culture system.

Vitrification of White-tailed Deer (Odocoileus virginianus) oocytes with sucrose or trehalose for in vitro maturation and fertilization.

Objective: Evaluate the White-tailed Deer (WTD) in vitro embryo production (IVP) and oocytes vitrified with Trehalose (TH) or Sucrose (SC).
 Design/methodology/approach: Total vitrified oocytes were placed into two different groups: TH (n=60) and SC (n=61). Samples were selected and analyzed for viability evaluation TH (n=5) and SC (n=5), nuclear status (NS) TH (n=4) and SC (n=5), Germinal Vesicle (GV), Metaphase I, or not evaluable (NE) after warming. In vitro maturation (IVM) was conducted for 36 h in supplemented TCM-199 medium. Immediately afterwards, oocyte NS was evaluated (n=88) [(GV, MI=immature), (MII=mature)]. In vitro fertilization (IVF) was performed in supplemented TALP medium for 24 h using frozen WTD semen (3x106 sperm/mL), NS was classified [Fertilized (F), Not fertilized (NF), or NE].
 Results: After warming, viability for the TH group (n=5) was 60% versus 40% for SC group (n=5), however, oocytes in both groups were immature (GV and MI stage). For IVM, NS evaluations of the TH group (n=38) revealed no maturation versus 2% in the SC group (n=50) (MII stage=matured). IVF evaluations for the TH group (n=10) revealed no fertilization compared to 20% in the SC group (n=5). A statistical difference (p>0.05) was not found between the TH and SC groups.
 Limitations on study/implications: White-tailed Deer in vitro embryo production is not well documented.
 Findings/conclusions: Future research with a larger number of WTD oocytes is needed for further evaluation of oocyte vitrification IVP techniques as a model for endangered cervids.

Effect of inducing accessory corpus luteum formation with gonadotropin-releasing hormone or human chorionic gonadotropin on the day of embryo transfer on fertility of recipient dairy heifers and lactating cows

The objective was to determine the effect of inducing an accessory corpus luteum (CL) using GnRH or human chorionic gonadotropin (hCG) on the day of in vitro-produced (IVP) embryo transfer (ET) on pregnancy per ET (P/ET) and calving/ET in dairy heifers and lactating cows. Dairy heifers (11–15 mo of age; n = 1,547) and lactating cows (n = 1,480) detected in estrus by tail chalk (d 0) were used as recipients. Before ET, the presence of a CL was evaluated by transrectal palpation from d 6 to 9 of the estrous cycle. Animals with a CL were randomly assigned to receive 1 of 3 treatments immediately before ET: control (no treatment; n = 1,009), GnRH (86 µg of GnRH; n = 1,085) and hCG (2,500 IU; n = 1,069). Embryos were implanted in the uterine horn ipsilateral to the ovary with a CL (fresh IVP embryos, n = 2,544; vitrified IVP embryos n = 545; slow-freezing IVP embryos, n = 74). Pregnancy diagnosis was performed on d 37 ± 3 of gestation by transrectal palpation. Pregnancy loss data and calving records were collected from the dairy farm management software. Treatment did not affect P/ET, calving/ET, or pregnancy loss either overall or within parity. When treatments inducing CL formation were combined (GnRH + hCG), heifers tended to have greater P/ET than controls (67.7 vs. 63.5%, respectively). Yet, calving/ET were similar. Response variables were also analyzed within embryo type and parity. For heifers receiving stage 6 (blastocyst) fresh IVP embryos, hCG had greater P/ET than controls (74.5 vs. 51.1%, respectively). In addition, GnRH tended to have greater P/ET than controls (67.8 vs. 51.2%, respectively). However, calving/ET in heifers receiving blastocyst fresh IVP embryos was similar among treatments. When only stage 7 (expanded blastocyst) fresh IVP embryos were considered, primiparous GnRH cows had greater P/ET (59.3 vs. 47.1%) and calving/ET (48.6 vs. 38.1%) than hCG. Moreover, hCG showed decreased calving/ET compared with controls in primiparous cows transferred with expanded blastocyst fresh IVP embryos. In summary, the effects of hCG or GnRH at ET on P/ET and calving/ET were inconsistent according to different embryo characteristics (e.g., embryo stage) and parity of recipients. Furthermore, treatment did not improve the overall fertility outcomes for recipient animals receiving IVP embryos.

Transmission of Mycoplasma bovis infection in bovine in vitro embryo production

Mycoplasma bovis (M. bovis) causes several costly diseases in cattle and has a negative effect on cattle welfare. There is no effective commercial vaccine, and antimicrobial resistance is common. Maintaining a closed herd is the best method to minimize the risk of introduction of M. bovis. Assisted reproduction is crucial in a closed herd to make genetic improvements. M. bovis has been found in commercial semen, and contaminated semen has been the source of disease in naïve dairy herds. The objective of this study was to evaluate M. bovis transmission in bovine in vitro embryo production (IVP) using several possible exposure routes. We used a wild-type M. bovis strain isolated from semen at a final concentration of 106 CFU/mL to infect cumulus–oocyte complexes, spermatozoa, and 5-day-old embryos. We also used naturally contaminated semen in fertilization. Blastocysts were collected on day 7–8 and zona pellucida (ZP)-intact embryos were either washed 12 times, including trypsin washes as recommended by the International Embryo Technology Society (IETS), or left unwashed. Washed and unwashed embryos, follicular fluids, maturation medium, cumulus cells, fertilization medium, and G1 and G2 culture media, as well as all wash media were analyzed using enrichment culture followed by real-time PCR detection of M. bovis. Altogether, 76 pools containing 363 unwashed embryos and 52 pools containing 261 IETS washed embryos were analyzed after oocytes, spermatozoa, or 5-day-old embryos were infected with M. bovis or naturally contaminated semen was used in fertilization. We could not detect M. bovis in any of the embryo pools. M. bovis was not found in any of 12 wash media from different exposure experiments. M. bovis did not affect the blastocyst rate, except when using experimentally infected semen. Contrary to an earlier study, which used a cell co-culture system, we could not demonstrate M. bovis in embryo wash media or tight adherence of M. bovis to ZP-intact embryos. Naturally infected semen did not transmit M. bovis to embryos. We conclude that by using our IVP system, the risk of M. bovis transmission via IVP embryos to recipient cows is very low.

In vitro embryo production in small ruminants: what is still missing?

In vitro embryo production (IVEP) is an extremely important tool for genetic improvement in livestock and it is the biotechnology that has grown the most recently. However, multiple ovulation followed by embryo transfer is still considered the leading biotechnology for embryo production in small ruminants. This review aimed to identify what is still missing for more efficient diffusion of IVEP in small ruminants, going through the IVEP steps and highlighting the main factors affecting the outcomes. Oocyte quality is essential for the success of IVEP and an aspect to be considered in small ruminants is their reproductive seasonality and strategies to mitigate the effect of season. The logistics for oocyte collection from live females is more complex than in cattle, and tools to simplify this collection system and/or to promote an alternative way of recovering oocytes may be an important point in this scenario. The heterogeneity of oocytes collected from growing follicles in live females or from ovaries collected from abattoirs remains a challenge, and there is a demand to standardize/homogenize the hormonal stimulatory protocols and IVM protocols for each source of oocytes. The use of sexed semen is technically possible, however the low market demand associated with the high costs of the sexing process prevents the routine use of this technique, but its higher availability is an important aspect aiming for greater dissemination of IVEP. New noninvasive approaches for embryo selection are key factors since the selection for transfer or cryopreservation is another difficulty faced among laboratories. Embryo selection is based on morphological traits, although these are not necessarily reliable in predicting pregnancy. Several issues described in this review must be considered by researchers in other to promote the diffusion of IVEP in small ruminants.

Outstanding Gir oocyte donors: How does individual factor affect in vitro embryo production efficiency?

The oocyte donor plays a pivotal role in bovine in vitro embryo production (IVP) success. The individual factor affects blastocyst/oocyte ratio and determine the existence of outstanding performing animals. The aim of this study was to assess the extent of individual factor effect to IVP efficiency, in a population of Gir oocyte donors. Extreme (high or low IVP efficiency based on blastocyst/oocyte ratio) animals were selected out of a population of 250 oocyte donors (1,734 observations) to form high (>0.48, n= 40), average (0.17-0.48, n= 168), and low (<0.17, n= 42) efficiency donor groups. Cumulus-oocyte complex indicators (total number, IVF-grade number, and IVF-grade/total ratio) were lower (p< 0.05) in high efficiency donors. The number of blastocysts per OPU was analyzed for highest performing bull, and an increase (p< 0.05) in high efficiency donors and a decrease (p< 0.05) in low efficiency donors were noticed, compared to average efficiency donors. The number of pregnancies obtained per OPU was affected (p= 0.017) by donor's efficiency (low: , average: , high: ), being 4.3-fold higher in high than in low efficiency donors. We conclude that producing embryos from high efficiency blastocyst/oocyte ratio donors increases blastocyst and pregnancy numbers by OPU, being an important indicator for donor selection in IVP programs.

The apoptotic inhibitor z-DEVD-fmk improves the viability and maturation rates of Iberian red deer oocytes while reducing apoptotic markers

The main drawback in developing in vitro embryo production (IVP) systems in wild species, such as the Iberian red deer, is that access to these animals is usually restricted, and long distances from the collection site to the laboratory are usually inevitable. Prolonged ovary storage is known to negatively influence the quality and developmental competence of the oocytes used for IVP. To overcome this issue, we evaluated the effect of adding a caspase-3 inhibitor, z-DEVD-fmk, to the in vitro maturation media to improve the quality and developmental potential of Iberian red deer oocytes. Oocytes were in vitro matured with and without z-DEVD-fmk, and the following parameters were analyzed: viability, early apoptosis, caspase-3 activity, DNA fragmentation, and relative abundance of mRNA transcript related to apoptosis. Moreover, oocyte maturation and blastocyst rates were also assessed. The results showed that z-DEVD-fmk decreased early apoptosis (inhibitor= 44.44 ± 3.6% vs. control= 60 ± 2.79%), DNA fragmentation (inhibitor= 57.83 ± 1.91% vs. control= 74.62 ± 1.91%), caspase-3 activity (inhibitor= 41.88 ± 3.42% vs. control= 67.10 ± 3.42%) and the relative abundance of TP53 and ITM2B transcripts, as well as increased the number of live (inhibitor= 41.48 ± 2.32% vs. control= 20 ± 1.8%) and in vitro-matured oocytes (inhibitor= 88.18 ± 1.99% vs. control= 74.01 ± 1.99%) rates. Nevertheless, the blastocyst production was not different between both experimental groups (inhibitor: 7.35 ± 2.30 vs. control: 13.77 ± 2.30). The supplementation of z-DEVD-fmk to the maturation medium improved the quality of Iberian red deer oocytes. Further research and alternative strategies are needed to evaluate if this inhibitor could still enhance the developmental potential of oocytes during prolonged ovarian transport.

Coculture with porcine luteal cells during in vitro porcine oocyte maturation affects lipid content, cortical reaction and zona pellucida ultrastructure.

In pigs, in vitro fertilisation (IVF) is associated with high polyspermy rates, and for this reason, in vitro embryo production (IVP) is still an inefficient biotechnology. Coculture with somatic cells is an alternative to improve suboptimal in vitro maturation (IVM) conditions. This study was conducted to test a coculture system of porcine luteal cells (PLC) and cumulus-oocyte complexes (COC) to improve oocyte metabolism. COC were matured in vitro with PLC. Oocyte lipid content, mitochondrial activity, zona pellucida (ZP) digestibility and pore size, cortical reaction and in vitro embryo development were assessed. Coculture reduced cytoplasmic lipid content in the oocyte cytoplasm without increasing mitochondrial activity. Although ZP digestibility and ZP pore number were not different between culture systems, ZP pores were smaller in the coculture. Coculture impacted the distribution of cortical granules as they were found immediately under the oolemma, and more of them had released their content in the ZP. Coculture with porcine luteal cells during IVM increased monospermic penetration and embryo development after IVF. The coculture of COC with PLC affects the metabolism of the oocyte and benefits monospermic penetration and embryo development. The coculture system with PLC could be an alternative for the conventional maturation medium in pigs.

A comparative study of oocyte between native cattle and buffalo species in Bangladesh: a qualitative and quantitative approach

In vitro embryo production (IVP) can help alleviating reproductive difficulties in cattle and buffalo. This study compared qualitative and quantitative features of native cattle and buffalo ovaries and oocytes to understand them comprehensively. In addition, it was conducted to pick the ideal ovaries for oocyte collection and to assess either maturation affects oocyte diameter or not. The ovaries were collected from the local slaughterhouses of Dhaka city, Bangladesh. Next, the quantitative parameters of ovaries were measured, the follicular fluid was aspired, the oocytes were classified, and the diameters of oocytes were determined in both pre and postmaturation. Here, the results revealed that the ovaries of cattle and buffalo differed significantly (p < 0.05) in weight, length, width, and depth regardless of the ovary's position and the corpus luteum (CL) condition. The numbers of normal and abnormal oocytes per ovary were 1.19 and 0.96 in cattle and 1.50 and 1.13 in buffalo, respectively. Moreover, a significant difference (p < 0.05) was observed between the oocytes' diameter in cattle and buffalo (cattle 85.64 ± 8.16 μm vs. buffalo 99.92 ± 8.65 μm) and before and after the maturation of oocytes within the same species. It may be concluded that the buffalo ovary was greater in size, and the oocyte was larger than the cattle ovary and oocyte. Any ovaries-irrespective of the ovary's position and the CL status-can be a good source of potential oocytes. Besides, the maturation process may ultimately affect oocytes' diameter increase.

Comparison of in vitro Maturation Media on Cattle Oocytes after in vitro Embryo Production

The study was conducted to compare differences in vitro maturation media and their effect on subsequent in vitro embryo production. The study procedures were approved by the agricultural research council animal ethics committee (Ref no: APAEC 2020/05) and the Tshwane university of technology animal research ethics committee (Ref no: AREC 2021/08/004). Follicular fluid containing a suspension of oocytes was retrieved from ovaries through aspiration and slicing techniques. The retrieved oocytes were graded and randomly matured into four media (TCM199, Vitromat Protect, and BO IVM®) groups for 22 h, evaluated for polar body extrusion, fertilized through their respective in vitro fertilization media, and evaluated for pronucleus status. Presumptive zygotes were further cultured in vitro in their respective media, incubated for either 18 or 96 h, and evaluated for cleavage rates. The data was subjected to an appropriate analysis of variance. Student's Least Significant Differences (t-LSD) was calculated at a 5% significance level to compare means of significant treatment effects. The BO-IVM® (57.4%), TCM 199 (55.6%), and VitroMat Protect® (54.0%) recorded higher results than EGF (26.9%) for polar body formation on oocytes (P<0.05). Vitrofert® medium recorded a higher percentage (43.3%) of total fertilization rate when compared to the other media (P<0.05). VitroCleave Plus® medium recorded a higher total cleavage rate (43.3%) as compared to the other media (P<0.05). In conclusion, TCM 199, BO-IVM®, and VitroMat Protect® media rendered higher results of oocyte polar body extrusion rates compared to the EGF medium. Vitrofert® medium recorded a higher percentage (43.3%) of total fertilization rate when compared to the other media. Higher embryo development (4-8 cells and total cleavage) rates were observed in VitroCleave Plus® medium. Overall, the introduced media (VitroMat Protect®) and its media suite successfully adapted to the laboratory environment in South Africa (SA) and can therefore be adopted for optimizing the in vitro Embryo Production (IVEP) of cattle oocytes.

Equine in vitro produced blastocysts: relationship of embryo morphology, stage and speed of development to foaling rate.

Information on factors associated with developmental competence of equine in vitro -produced (IVP) blastocysts is lacking. To determine the relationships of stage, grade, day of development, and specific morphological parameters of equine IVP blastocysts, to pregnancy and foaling rates. Photomicrographs of 316 IVP embryos with known pregnancy outcomes were scrutinised individually by four observers. Inter-observer variation was assessed, and pregnancy outcome evaluated in relation to day of blastocyst development and assigned grade and stage. Individual component analysis was performed to determine the association of specific morphological parameters with foaling rate. Overall pregnancy rate was 76.9% and foaling rate was 56.3%. The day of embryo development did not affect pregnancy rate but significantly affected foaling rate. Embryo stage did not affect foaling rate. Embryo grade affected foaling rate only for Day-9 embryos. Some morphological features in the bovine grading system did not predict outcome in equine IVP embryos. Significant individual parameters differed between Stage 5 and Stage 6 equine blastocysts. Day of blastocyst development is the major factor related to foaling rate for equine IVP embryos. Notably, there was no effect of embryo stage on foaling rate and no evidence that prolonging culture until embryos advance in stage increases foaling rate. The standard bovine grading system is not directly applicable to equine IVP embryos; equine-specific staging and grading systems are proposed. This information will allow laboratories to identify embryos with the highest developmental competence. Use of the proposed systems will increase consistency in embryo assessment among laboratories.

Effects of the temperature-humidity index on conception rates in Holstein heifers and cows receiving in vitro-produced Japanese Black cattle embryos.

We investigated the effect of the temperature-humidity index (THI) on the conception rate (CR) in Holstein heifers and cows receiving in vitro-produced (IVP) Japanese Black cattle fresh embryos. IVP embryos were transferred to Holstein heifers (n = 1,407) and cows (n = 3,189) on 245 commercial farms. The monthly average ambient temperature (AT) and THI ranged from 4.7 to 29°C and 41 to 81, respectively; both were the highest in August. The monthly CR ranged from 16.3% to 46.7% in cows and 23.8% to 74.1% in heifers. The CR of heifers was unaffected by THI, AT, or the month of embryo transfer. However, these parameters affected the CR of cows. The CR at THI values of 61-65 and 71-75 was greater than that at THI > 75, whereas other THI values had no effect. The CR at temperatures > 25°C was lower (P = 0.008) than that at temperatures of 15-20°C and 20-25°C. Moreover, the CR was lowest (P = 0.003) in July. THI and parity (P = 0.057 and P = 0.001, respectively) and AT and parity (P = 0.019 and P = 0.001, respectively) showed significant effects on CR; however, there was no interaction between these two factors. In conclusion, AT > 25°C and THI > 75 adversely affect the CR outcome in cows but not in heifers.

Transvaginal ultrasound-guided oocyte retrieval in cattle: State-of-the-art and its impact on the in vitro fertilization embryo production outcome.

Transvaginal ultrasound-guided oocyte retrieval (commonly called OPU) and in vitro embryo production (IVP) in cattle has shown significant progress in recent years, in part, as a result of a better understanding of the full potential of these tools by end users. The combination of OPU and IVP (OPU-IVP) has been successfully and widely commercially used worldwide. The main advantages are a greater number of embryos and pregnancies per unit of time, faster genetic progress due to donor quick turn around and more elite sires mating combinations, larger spectrum of female age (calves, prepuberal, heifer, cow) and condition (open, pregnant) from which to retrieve oocytes, a reduced number of sperm (even sexed) required to fertilize the oocytes, among other benefits. OPU-IVP requires significant less donor preparation in comparison to conventional embryo transfer (<50% of usual FSH injections needed) to the extent of no stimulating hormones (FSH) are necessary. Donor synchronization, stimulation, OPU technique, oocyte competence, embryo performance, and its impact on cryopreservation and pregnancy are discussed.

Factors Affecting the Efficiency of In Vitro Embryo Production in Prepubertal Mediterranean Water Buffalo.

Embryos from prepubertal water buffalo can be produced using laparoscopic ovum pickup (LOPU) and in vitro embryo production (IVEP). However, to date, it is unclear what factors and environmental conditions can affect LOPU-IVEP efficiency in prepubertal animals, especially buffalo. In this study, we explored the impact of season, age and individual variation among female donor animals, as well as the effect of the sire used for in vitro fertilization. Donor animals between 2 and 6 months of age were stimulated using gonadotropins prior to LOPU, which was performed at two-week intervals. Following in vitro maturation and fertilization, the resulting embryos were then cultured to the blastocyst stage until they were either vitrified or transferred into recipient animals. The number of follicles available for aspiration and embryo development rates was stable throughout the year. As animals became older, there was a slight trend for fewer COCs recovered from LOPU and better embryo development. There was a large individual variation in both ovarian response and the developmental competence of oocytes among donors. The bull used for fertilization also had a significant impact on embryo development. Upon embryo transfer, pregnancy rates were not affected by the number of embryos transferred per recipient. The best pregnancy rates were achieved when transferring blastocysts, compared to compact morula or hatched blastocysts. Finally, vitrification had no effect on pregnancy rate compared to fresh embryos.

Fitness of calves born from in vitro-produced fresh and cryopreserved embryos.

In cattle, vitrified/warmed (V/W) and frozen/thawed (F/T), in vitro-produced (IVP) embryos, differ in their physiology and survival from fresh embryos. In this study, we analyzed the effects of embryo cryopreservation techniques on the offspring. IVP embryos cultured with albumin and with or without 0.1% serum until Day 6, and thereafter in single culture without protein, were transferred to recipients on Day 7 as F/T, V/W, or fresh, resulting in N = 24, 14, and 13 calves, respectively. Calves were clinically examined at birth, and blood was analyzed before and after colostrum intake (Day 0), and subsequently on Day 15 and Day 30. On Day 0, calves from V/W and F/T embryos showed increased creatinine and capillary refill time (CRT) and reduced heartbeats. Calves from F/T embryos showed lower PCO2, hemoglobin, and packed cell volume than calves from V/W embryos while V/W embryos led to calves with increased Na+ levels. Colostrum effects did not differ between calves from fresh and cryopreserved embryos, indicating similar adaptive ability among calves. However, PCO2 did not decrease in calves from V/W embryos after colostrum intake. Serum in culture led to calves with affected (P < 0.05) temperature, CRT, HCO , base excess (BE), TCO2, creatinine, urea, and anion gap. On Day 15, the effects of embryo cryopreservation disappeared among calves. In contrast, Day 30 values were influenced by diarrhea appearance, mainly in calves from V/W embryos (i.e., lower values of TCO2, HCO , and BE; and increased glucose, anion gap, and lactate), although with no more clinical compromise than calves from fresh and F/T embryos. Diarrhea affected PCO2 and Na+ in all groups. Embryo cryopreservation, and/or culture, yield metabolically different calves, including effects on protein and acid-base metabolism.

Direct transfer of a single frozen-thawed in vitro-produced embryo to avoid twin pregnancy in dairy cows.

Fertility of cow receiving fixed-time artificial insemination (FTAI) was compared with cow in which a single frozen-thawed in vitro-produced (IVP) embryo was fixed-time transferred (FTET) to avoid twin pregnancy. The study population was comprised of 596 lactating dairy cows synchronized for oestrus: 440 were fixed-time inseminated (AI cows), and 156 were given GnRH treatment at the time of embryo transfer (ET cows) 8 days post-oestrus. Of the 596 cows, 235 (39.4%) became pregnant: 175 (39.8%) AI cows and 60 (39.8%) ET cows. Twin pregnancy was recorded in 16% of the AI pregnant cows (28/175), whereas no ET cows had twins (0/60). Significant interaction (p < .01) was observed between breeding technique (FTAI vs FTET) and repeat (RB) or not repeat breeding (NRB) for the likelihood of pregnancy. This meant that using RB AI cows as reference, the odds ratio for pregnancy in RB ET cows was 2.2 (p=.04). In conclusion, transfer of a frozen IVP embryo proved useful to prevent the risk of twin pregnancy without affecting fertility.

In Vitro Embryo Production in Water Buffalo

In vitro embryo production (IVEP) is a promising tool with many applications in producing calves from genetically superior animals desired for propagation and in the conservation and revival of endangered species. The techniques of IVEP were adopted from cattle and refined to suit the water buffalo requirements. From the collection of ovaries from a local abattoir and the collection of oocytes by ovum pick up from live animals, gamete storage, collection techniques, handling of ovaries and oocytes to keep the viability and developmental competence, selection of oocytes to the type of culture media and in vitro culture condition, and treatment of the sperm cells for in vitro fertilization are all-important components of the process that requires careful and precise action to ensure success. Trials on intracytoplasmic injection, the use of sex-sorted sperm cells as a tool for producing sex-predetermined embryos, and the somatic cell nuclear transfer are methods that can be used to produce embryos in vitro. This paper provides the important considerations involved in the production of healthy live calves out of in vitro-produced water buffalo embryos.

Comparison of the efficiency of progesterone, ketoprofen, or GnRH administration on the day of fixed-time transfer (FTET) of in vitro produced (IVP) embryos in suckled crossbred beef cows

ABSTRACT The objective is to treat cows receiving IVP embryos with injectable progesterone, ketoprofen, and gonadorelin on the day of FTET and to compare the reproductive efficiency of untreated cows with that of treated cows, using pregnancy per ET as the response variable. 32 oocyte donor cows were used and 392 embryo transfers were performed in multiparous cows. Recipient cows were synchronised with the protocol: on day zero (d0), an intravaginal progesterone device was inserted (1.0 g P4), and 2.0 mg estradiol benzoate (EB;im) was administered; on d8, the P4 device was removed, and 150 µg of cloprostenol was administered; on d9, 1.0 mg EB was administered; and on d17, embryo transfers were performed. On d17, recipient cows were randomly assigned to one of four groups: those treated with injectable progesterone (iP4G, 150 mg; n = 84), ketoprofen (KetoprofenG, 1000 mg; n = 104), gonadorelin (GonadorelinG, 100 µg; n = 80), or the control (untreated) group (NTG, n = 124). Pregnancy diagnosis was performed 35 days following embryo transfer, showing pregnancy per FTET of 42.8, 57.6, 55.0, and 43.5% for iP4G, KetoprofenG, GonadorelinG, and NTG, respectively. It is concluded that ketoprofen improves FTET pregnancy rates when compared to injectable progesterone and control group. Gonadorelin demonstrated comparable efficacy to ketoprofen.

In vitro production of buffalo embryos in Colombia – results of the addition of growth factors and antioxidants to defined culture medium

To increase the success rate of the in vitro production (IVP) system of embryos in buffalo, it is necessary to improve parameters. This field requires more research since its results are low compared to cattle. In this study carried out in Colombia, a modification of the methodology to produce buffalo IVP embryos is described, and 24 non-lactating females, aged 4 to 11 years old were included. Follicular wave was synchronised by ablation of the follicles within the ovary, seven days later all follicles were aspirated to obtain oocytes for maturation, fertilisation and embryo production. For all culture media used, animal protein was avoided. Culture media were supplemented with growth factors and other molecules associated with embryonic development. Special care was taken in adding supplements to protect the zygotes against the harmful effect of oxygen free radicals in the culture medium. Two aspiration series were performed 15 days apart. The average number of oocytes obtained by aspiration was 11.33, the number of blastocysts was 3.05, and the obtained blastocyst production rate (36.2%) was higher than literature reports for this species; we hypothesise that this increase is associated with the supplements added. It was possible to observe a female that produced 50 oocytes. Subsequent research is needed to evaluate the effect of the medium on embryo quality and pregnancy rates. A cost analysis of the new proposed medium is required, since all supplements must be imported and handled with a cold chain of up to -80oC, making it more expensive and restricting widespread use. Finally, it is shown that with three embryos per aspiration, at least one pregnancy can be successful after the aspiration, which is one of the objectives of the IVP system.

Metabolic signature of spent culture media shows lipid metabolism as a determinant of pregnancy outcomes.

In the present study, we investigated the spent culture media of in vitro produced (IVP) bovine embryos which did (group Pregnant) or did not (group Non-pregnant) establish pregnancy after transfer. For that purpose, IVP embryos on D5 were transferred to individual droplets for the last 48h of culture. Embryos at the blastocyst stage were then transferred to synchronized recipients, while respective culture media drops were collected and evaluated individually. The list of metabolites present in spent culture media was obtained by electrospray ionization mass spectrometry (ESI-MS) and analysed with Metaboanalyst® to characterize the metabolic profile of each group. The spectrometric analysis showed that pathways related to lipid metabolism, particularly fatty acids degradation via beta-oxidation, were more present in the Pregnant group whereas no significant pathway was identified in the group Non-pregnant. By using this method, we were able to identify a metabolic signature in culture media that allows for a better comprehension of preferential metabolic routes taken by the most viable embryos. These findings offer great insights into the biochemistry of embryo development and reveal a potential target for the development of better-quality IVP systems, as well as tools to identify bovine embryos with greater chances to establish and maintain pregnancy.