Fert-TALP Medium Research Articles

Prolonged incubation of frozen-thawed equine spermatozoa for invitro fertilization: A preliminary study using low temperature and INRA96 medium.

A protocol for conventional invitro fertilization (IVF) in horses using fresh semen has been described, using a prolonged incubation in FERT-TALP medium (22 h) at 38.2°C in the presence of penicillamine, hypotaurine and epinephrine (PHE). Our work aimed to develop a protocol that maintains quality parameters in frozen-thawed equine spermatozoa incubated for 22 h in the presence of PHE using different media (FERT-TALP and INRA96) and incubation temperatures (30 and 38.2°C). Twelve frozen ejaculates from four stallions were thawed and then incubated in either FERT-TALP or INRA96 with PHE at 30 or 38.2°C for 22 h. Following incubation, total motility (TM), progressive motility (PM), viability and acrosome integrity were evaluated. The results showed that TM was significantly higher (p < .001) at 30°C in both media, while PM was higher for INRA96 at 30°C compared to 38°C (p < .05). Moreover, INRA96 at 30°C exhibited higher sperm viability and acrosome integrity (p < .001) compared to the other experimental groups. These preliminary results suggest that incubating thawed equine spermatozoa at 30°C with PHE in INRA96 successfully maintains motility, viability and acrosome integrity in equine spermatozoa, indicating its potential use for conventional equine IVF.

Evaluation of epididymal and frozen sperm to produce goat embryos through in vitro fertilization

Preservation of the livestock genetic makeup could be performed by application of in vitro fertilization (IVF) using spermatozoa collected from cauda epididymis. Therefore, this study as sessed the development of goat embryos following IVF of spermatozoa collected from cauda epididy mis compared to those kept frozen. Oocytes were in vitro cultured in a maturation medium for 24 hours at a temperature of 38.5°C, 5% CO2, and 95% humidity. Following maturation, the oocytes (n = 370) were in vitro fertilized by fresh epididymal sperm (G1) and G2 frozen-thawed sperm, and the Fert-TALP medium was used as the IVF medium. In addition, in vitro developed embryos were cultured in GT-L medium. The fertilization rate, percentage of morula, and blastocytes (p&lt;0.01) were significantly higher in oocytes inseminated with epididymal than in frozen sperm. In conclusion, in vitro embryo production of goat oocytes may be successfully performed using epididymal spermatozoa for IVF.

13 Generation of SLICK beef cattle by embryo microinjection: A case report

Due to climate change, cattle can experience heat stress more frequently in traditionally temperate, non-tropical environments. Because of this and the fact that most of the world’s inefficient cattle reside in tropical zones, we set out to demonstrate our ability to adapt Angus animals to heat stress through a single gene edit in the prolactin receptor (PRLR). We selected PRLR, because it is known that the SLICK phenotype results from 1 of 3 different mutations in PRLR and therefore is related to heat stress regulation. The overall project was initiated through a partnership between Acceligen and BluePrint Genetics (BPG). BPG harvested 1415 oocytes from a total of 9 ovum pickup rounds from 7 different Angus donors. These oocytes were graded, selected, and put in maturation medium for shipping to Acceligen. Matured oocytes were fertilized in Fert-TALP medium and co-incubated with frozen/thawed semen from 3 different reproductive certified Angus bulls. Consequently, targeted editing was done on a single cell in the zygotes 12h after fertilization by introduction of guide (g)RNA/Cas9 (250ng μL−1 of each) through intracytoplasmic microinjection. Then, treated zygotes (n=1341) were cultured in synthetic oviductal fluid with amino acids (SOFaa) culture medium under a controlled atmosphere until on Day 6, when 277 (20%) grade 1 embryos were selected and 39 returned to Blueprint Genetics for transfer. Because the number of available recipients was limited, only 4 rounds of fresh embryo transfer were completed, and the remaining embryos (n=190) were vitrified for future transfers. Pregnancy checks by ultrasound on Day 30 revealed a 23% (9/39) rate of pregnancy, which decreased to 13% (5/39) by Day 60. In total, 4 animals reached term and delivered healthy calves. Genetic testing of the PRLR target site was done by amplicon sequencing, which showed 3 edited SLICK animals (75%) and one wild-type. Thus, the SLICK zygote editing by microinjection was demonstrated to be an efficient method to produce bovine beef animals and is currently being prepared for regulatory review in multiple countries and commercialization.

80 Energetic substrate availability affects the metabolome profile in bovine sperm

Bovine spermatozoa are specialised cells that require high ATP production for flagellar movement and other physiological events necessary for fertilization. Glycolysis and oxidative phosphorylation (OXPHOS) are the most studied energy pathways in sperm cells and involve metabolites such as glucose, pyruvate, and lactate. Although glycolysis has been described as the preferential pathway for ATP generation in bovine spermatozoa, other metabolites may also be used, leading to distinct metabolome profiles. Thus, the objective of this work was to characterise the metabolome profile of culture media derived from sperm cells incubated in the presence of different energy substrates for ATP production. For that, a semen straw from one bull (n=3) previously tested for IVF was thawed and motile sperm were separated by Percoll gradient, washed and resuspended in FertTalp medium (FT) without capacitator agents (Parrish et al. 1989 Biol. Reprod. 41, 683–699) to a final concentration of 30×106cells mL−1. Then, samples were centrifuged and resuspended in 5 different groups: positive control (PC, FT supplemented with 2mM glucose, 0.2mM pyruvate, and 11mM lactate); negative control (NC, FT without energy substrates); Glu (FT and 3.5mM glucose); Pyr (FT and 0.11mM pyruvate), and Lac (FT and 5.5mM lactate). Samples were incubated at 38.5°C, 5% CO2 in high humidity for 15 and 45min. After incubation, samples were centrifuged and supernatant was collected and analysed by Raman spectroscopy as previously described (Santos et al. 2015 Biomed. Opt. Express 6, 2830–2839; https://doi.org/10.1364/BOE.6.002830). Data were preprocessed and submitted to principal component (PCA) and loading plot analysis (LP) by using Minitab software (Minitab Ltd.). The results showed that after 15min of incubation, the metabolic profiles were similar for the PC, Glu, and Lac groups, suggesting that they present similar metabolic activity. NC and Pyr were a separate cluster, indicating that pyruvate is not metabolized through OXPHOS in this phase. LP analysis comparing Glu and Pyr groups indicated phosphatidylserine, phenylalanine, DNA/RNA, and lipids as the most distinct metabolites. After 45min, PC and Pyr had a similar metabolome profile, whereas NC, Glu, and Lac clustered together, suggesting that for long-period incubation OXPHOS takes place as the preferential pathway for energy production in bovine sperm cells. At this time, the comparison of Glu versus Pyr revealed phosphatidylserine, proline, phenylalanine, carboxylic acid, DNA/RNA, proteins, and lipids as the most different metabolites between groups. Based on these results, we hypothesised that the glycolysis to OXPHOS transition may be a consequence of the depletion of glycolytic enzymes, leading the sperm cells to use distinct pathways for long-term maintenance of ATP production. In conclusion, our data showed that the metabolome profile of bovine spermatozoa varies according to the period of incubation and substrates availability for energy production. However, more studies are necessary to characterise the ability of these metabolites to maintain sperm motility and viability. This research was funded by FAPESP 2017/18384-0.

Assessment of binder of sperm protein 1 (BSP1) and heparin effects on in vitro capacitation and fertilization of bovine ejaculated and epididymal sperm.

The present study evaluated the effect of binder of sperm protein 1 (BSP1) and/or heparin on in vitro bovine capacitation and fertilization rates using epididymal and ejaculated bovine sperm. Frozen-thawed sperm were selected and used in the following treatments. Control group: Fert-TALP medium without heparin; heparin (HEP) group: Fert-TALP with heparin (10 UI/ml); BSP1 group: Fert-TALP medium with BSP1 (10 µg/ml for ejaculated sperm; 40 µg/ml for epididymal sperm); HEP + BSP1 group: Fert-TALP medium with heparin (5 UI/ml) and BSP1 (5 µg/ml for ejaculated sperm; 20 µg/ml for epididymal sperm) and determined in vitro capacitation rates in different interval times (0, 15, 30 and 60 min) using the chlortetracycline fluorescence (CTC) method. Also, we evaluated the development rates of oocytes fertilized with ejaculated or epididymal sperm into the same treatments. Capacitation was greater and faster when ejaculated sperm were treated for 60 min with heparin compared with other treatments. However, developmental rates were similar in all treatments. For epididymal sperm, the treatments with BSP1 presented higher capacitation and fertilization rates compared with heparin (P < 0.05). The effects of heparin + BSP1 on capacitation and developmental rates did not cause any increase in capacitation or blastocyst rates compared with other groups for ejaculated or epididymal sperm. In conclusion, this study confirmed that either BSP1 and heparin can be used as capacitator agents for bovine ejaculated sperm during IVF. However, BSP1 seems to be more efficient compared with heparin for epididymal sperm. Furthermore, BSP1 and heparin have no synergic effects on sperm capacitation.

In Vivo and In Vitro Dromedary Camel Embryos’ Production under Egyptian Conditions

The aim of the present study was to compare between in vivo and in vitro production of dromedary camel embryos under Egyptian conditions. At in vivo experiment, three female camels (donors) were treated with a one-shot injection of 3000 IU PMSG, followed by induction of ovulation treatment (i.m. administration of 5000 IU hCG). Natural mating was performed after 8 days from the superovulation treatment. Embryos were flushed via non-surgical collection after eight days of animal mating. For in vitro experiment the oocytes were retrieved by slicing ovaries collected from the slaughterhouse. Recovered oocytes were examined before and after IVM culture for grading and investigating cytoplasmic maturation. Only good-quality oocytes were selected (n=72). Cumulus oocytes complexes (COCs) were incubated at 38.5 oC, 5% CO2 and 95% humidity for 40 hours. Matured COCs were inseminated with frozen-thawed semen (3x106 spermatozoa/mL) in the fert-TALP medium at 38.5 oC, 5% CO2 and 95% humidity for 18 hours. Results showed that the in vivo embryos recovery rate was 74.92%. The hormonal treatment had no significant effect on the activity of ovaries regarding ovarian side. For in vitro experiment the mean oocyte yield was 9.83 COCs per ovary and the recovery rate (%) was 37.5%. The maturation rate of dromedary oocytes in vitro was 72.72%, while the fertilization rate was 11.5%. In conclusion, this study showed that in vivo embryo production can be considered as an effective tool for embryo in dromedary camels when compared to embryo production in vitro.

Bovine oviductal fluid (bOF) collected in the follicular or luteal phase of the estrous cycle exerts similar effects on ram sperm kinematics and acrosome reactivity in vitro

This study examined the effects of bovine oviductal fluid (bOF) obtained during the follicular or luteal phase of the estrous cycle on ram sperm kinematics, capacitation status and plasma membrane (PM) integrity at various time points during the 24-h incubation period. Fresh ram spermatozoa were selected using the swim-up technique and then incubated separately with either follicular phase (FbOF) or luteal phase (LbOF) bovine oviductal fluid added to Fert-TALP medium (positive control - POSControl) or in Fert-TALP medium without capacitating agents (negative control - NEGControl) at 38 °C under 5% CO2. Incubation with FbOF or LbOF for 2 h and 4 h promoted an increase (P < 0.05) in most of the sperm motility parameters as compared with the NEGControl group, and bOF-induced changes in sperm kinematics were similar (P > 0.05) to those seen in the POSControl group. After 6 h of incubation, the stimulatory effect of FbOF or LbOF on ram sperm kinematics was no longer observed (P > 0.05). Sperm PM integrity was not affected (P > 0.05) by incubation in bOF-supplemented media or in absence of capacitating factors (NEGControl). Although neither FbOF nor LbOF had any effect on sperm capacitation rates, the proportion of acrosome-reacted spermatozoa was greater (P < 0.05) for bOF-containing media compared with the NEGControl group during the long incubation periods (18 h and 24 h). In conclusion, bOF from either follicular or luteal phase of the estrous cycle enhances ram sperm motility for up to 4 h and the rate of acrosome reaction after long (18–24 h) incubation periods without affecting sperm viability.

171 Effect of progesterone and prolactin on the developmental competence of bovine oocytes during the terminal phase of in vitro maturation

To date, considerable progress has been achieved in in vitro production (IVP) technologies in cattle; however, developmental potentials of oocytes matured in vitro remain low compared with in vivo-matured oocytes. Thus, a better understanding of different aspects of oocyte maturation may allow us to increase the embryo development rate. Our study was aimed to assess the effects of progesterone (P4) and prolactin (PRL) on the bovine oocyte developmental competence. Bovine cumulus-enclosed oocytes (CEO) were matured using either one-step or two-step maturation conditions. For the one-step protocol, CEO were cultured for 24h in TCM-199 supplemented with 10% fetal calf serum (FCS), 10μg mL−1 porcine FSH, and 10μg mL−1 ovine LH (standard medium). For the two-step procedure, CEO were first cultured for 16h in the standard medium (n=1263) and then transferred to 1 of 3 experimental media and cultured for additional 8h in either absence or presence of either P4 (50 ng mL−1) or bovine PRL (50ng mL−1). The 3 media tested in the two-step maturation were (1) TCM-199 containing 10% FCS (group 1), (2) TCM-199 containing 3mg mL−1 BSA (group 2), or (3) Fert-TALP medium supplemented with 6mg mL−1 BSA (group 3). Fert-TALP was selected because it can potentially be used throughout maturation and fertilization. Following in vitro maturation, all oocytes underwent an IVF/in vitro culture procedure as described previously (Singina et al. 2014 Reprod. Fertil. Devel. 26, 154). The embryo development was evaluated at Days 2 and 7 for cleavage and blastocyst rates. In addition, obtained blastocysts were fixed, and the total cell number and the level of apoptosis were determined using 4′,6-diamidino-2-phenylindole (DAPI) and TUNEL staining. The data from 4 to 5 replicates (113-159 oocytes per treatment) were analysed by ANOVA. For oocytes matured for 24h in the one-step culture, the cleavage rate, blastocyst rate, total cell number, and apoptotic nuclei per blastocyst were 66.1±1.1, 23.7±2.0, 71.4±9.1, and 4.8±1.2%, respectively. For the two-step culture, the cleavage rate did not differ from that of the one-step culture system, ranging from 64.8 to 76.5%. Also, no effects of the two-step systems were observed on total cell number (63.0-78.8) or the proportion of apoptotic nuclei (3.3-5.3%) at the blastocyst stage. The culture of CEO in group 1 (without the supplements) had a reduced blastocyst rate (17.4±0.4%; P&amp;lt;0.05) compared with the standard one-step maturation group, and the addition of P4 (but not PRL) improved the blastocyst yield (P&amp;lt;0.05). Furthermore, when P4 (but not PRL) was added to group 2 and group 3 media, blastocyst rates increased significantly (32.9±3.1 and 32.8±2.7%, respectively) compared with those of the one-step group (P&amp;lt;0.05), but did not differ from those of untreated groups 2 and 3 (26.2±2.7 and 30.0±3.0%, respectively). Our data indicate that P4 supplementation during the terminal phase of two-step IVM can enhance the developmental competence of bovine oocytes and that the nature of this effect depends on the composition of IVM medium, whereas no effect of PRL supplementation was observed. The study was supported by RFBR (No. 17-29-08035).

Supplementation of 17β-estradiol and progesterone in the co-culture medium of bovine oviductal epithelial cells and ovine spermatozoa reduces the sperm kinematics and capacitation

This study investigated the effect that bovine oviductal epithelial cell (BOEC) and ovine spermatozoa co-culture exposed to different hormonal environments had on ram sperm function over the course of a 24-h incubation period. Ram cooled-stored spermatozoa were selected by swim-up and then co-cultured separately for 24 h at 38.5 °C under 5% CO2 with either: (1) Fert-TALP medium (positive control [POSControl]), (2) Fert-TALP medium supplemented with 17β-estradiol (E2) and progesterone (P4) at concentrations similar to follicular phase (Follicular NEGControl), (3) Fert-TALP medium supplemented with E2 and P4 concentrations similar to luteal phase (Luteal NEGControl), (4) BOEC cultured in the same medium as that of the Follicular NEGControl group (Follicular BOEC group), or (5) BOEC cultured in the same medium as that of the Luteal NEGControl group (Luteal BOEC group). The sperm kinematics, capacitation status, and plasma membrane (PM) integrity were evaluated at different intervals. Sperm PM integrity was not affected (P ˃ 0.05) by BOEC co-culture, regardless of the phase of the estrous cycle. After 4 h of incubation, the Luteal BOEC group presented lower (P < 0.05) progressive motility and total motility than the Luteal NEGControl group while the Follicular BOEC group showed lower (P < 0.05) velocimetric parameters and progressive motility than the Follicular NEGControl group. Throughout the incubation period, both BOEC co-culture groups showed a decrease (P < 0.05) in their capacitation rate in comparison to the POSControl group. Conversely, the Luteal BOEC group presented a higher (P < 0.05) non-capacitated rate than both the POSControl and Luteal NEGControl groups. In conclusion, BOEC co-culture with ovine spermatozoa at either the follicular or luteal phase decreases sperm kinematics and delays sperm capacitation.

In Vitro Maturation and Fertilization of Donkey Oocytes

Many wild and domestic donkey breeds are now at risk of extinction, therefore there is an urgent need for genetic conservation of these breeds. The development of in vitro conditions that support oocyte maturation, fertilization, and embryo development may improve in vitro production of donkey embryos. This work was designed to identify the optimal condition for in vitro production of donkey embryos. Two experiments were conducted. In experiment 1, grade 1 and 2 cumulus-oocyte complexes were subjected to in vitro maturation in the following media: tissue culture medium 199 (TCM199), Christopher Rozenkrans 1aa medium (CR1aa), Christopher Rozenkrans 2aa medium (CR2aa), potassium simplex optimized medium (KSOMaa), centrifuged follicular fluid (CFF), or non-CFF from preovulatory follicles. In experiment 2, in vitro–matured donkey oocytes were subjected to in vitro fertilization (IVF) in Fert-TALP medium supplemented with 0%, 25%, 50%, 75%, or 100% CFF. After fertilization, the cleavage rates and embryo development were assessed across groups. The results of experiment 1 revealed that non-CFF and KSOMaa significantly increase cumulus cell expansion. The percentage of oocytes reaching the metaphase II stage nuclear maturation was significantly higher for donkey oocytes matured in non-CFF. Also, the percentage of degenerated oocytes was lower for donkey oocytes matured in vitro in non-CFF compared with those in TCM199, CR1aa, CR2aa, KSOMaa, or CFF. In experiment 2, IVF of in vitro–matured donkey oocytes using 100% CFF as IVF medium produced a significantly higher fertilization rate with respect to the number of fertilized oocytes extruding the second polar body. In conclusion, non-CFF from preovulatory follicles gave the highest cumulus cell expansion and nuclear maturation rates for in vitro–matured donkey oocytes. Hundred percent CFF was the best medium for IVF of matured donkey oocytes; however, cleavage rate and embryo development are still very low and need further investigations.

Effects of different activation protocols on cleavage rate and blastocyst production of caprine oocytes.

The present study was undertaken to assess the effect of different chemical activators along with 6-DMAP on in vitro matured caprine oocytes. From 4332 ovaries, 14235 cumulus oocyte complexes (COCs) were collected which were matured in TCM-199 medium containing follicle stimulating hormone (FSH) (5 µg/ml), Leutinizing hormone (LH) (10 µg/ml), oestradiol-17β (1 µg/ml) supplemented with 10% fetal bovine serum, 10% follicular fluid and 3 mg/ml bovine serum albumin (BSA) at 38.5°C and 5% CO2 in an incubator under humidified air for 27 h. In group 1 (control), 3117 in vitro matured oocytes were co incubated with sperms for 18 h in ferttalp medium. In group 2, 3563 in vitro matured oocytes were activated with 7% ethanol for 5-7 min followed by treatment with 2.0 mM DMAP for 4 h in mCR2aa medium. In group 3, 3109 in vitro matured oocytes were activated with 5 μM ionomycin for 5-7 min followed by treatment with 2.0 mM DMAP for 4 h in mCR2aa medium. In group 4, 3455 in vitro matured oocytes were activated with 5 μM calcium ionophore for 5-7 min followed by treatment with 2.0 mM DMAP for 4 h in mCR2aa medium. Oocytes were cultured in 50 µL drops of research vitro cleave (RVCL) medium for embryo development. The cleavage rate, morula and blastocyst production in group 1, 2, 3 and 4 were 26.07 ± 2.37%, 14.91 ± 2.91 & 1.45 ± 0.71%, 49.57 ± 3.79%, 20.07 ± 2.38% & 5.29 ± 1.42%, 50.18 ± 3.59%, 15.26 ± 2.87% & 1.85 ± 0.72% and 80.26 ± 2.30%, 35.33 ± 2.67 & 7.10 ± 0.89%, respectively. These results indicated that the activation of in vitro matured oocytes by 5 μM calcium ionophore for 5-7 min followed by treatment with 2.0 mM DMAP for 4 h is most favorable for parthenogenetic caprine embryos production.

Effect of Fetal Calf Serum and Mid Estrus Cow Serum on In Vitro Maturation and Fertilization of Oocytes from Crossbred Cattle in the Tropics

The objective of the study was to examine the effect of fetal calf serum (FCS) and mid estrus cow serum (ECS) in culture media on in vitro maturation (IVM) and in vitro fertilization (IVF) of oocytes from crossbred cattle in the tropics. Oocytes from abattoir ovaries were cultured in three different maturation media at 39oC temperature, 5% CO2 tension with maximum humidity for 24 h. TCM-199 containing 25mM HEPES, 1mM glutamine L, 2.2mg/mL sodium bicarbonate, antibiotics, 22 µg/mL pyruvate, 1µg/mL estradiol-17β, 0.5µg/mL FSH and 0.06 IU hCG without serum supplement used as treatment-1 and the above media were further supplemented with 10% FCS as treatment-2. In treatment-3, instead of 10% FCS, 20% heat inactivated ECS (serum collected in mid estrum) was used as serum supplement. Oocytes with maximum degree of cumulus expansion were selected as matured and used for further IVF studies using frozen semen. The IVF medium consisted of Fert-TALP medium supplemented with 1µM epinephrine, 10µM hypotaurine, 20µM pencillamine and 0.56µg/ml heparin. Culture conditions set for IVF were 39oC temperature, 5% CO2 tension with maximum humidity. Oocytes showing sperm penetration evidence like presence of enlarged sperm head, male pronuclei with its accompanying sperm tail in the cytoplasm, oocytes with two pronuclei and a clear second polar body but without a sperm tail were considered as fertilized. Significantly higher result of cumulus expansion percentage (p&lt; 0.05) was observed when oocytes matured in media supplemented with FCS as compared to other two treatments. There was no influence of source of serum in maturation media on further IVF of matured oocytes in this study. Int J Appl Sci Biotechnol, Vol 4(3): 308-310

In Vitro Fertilizability of Oocytes Using Fresh, Frozen and Epididymal Spermatozoa from Crossbred Bulls in the Tropics

The objective of the study was to examine the fertilizability of bovine follicular oocytes from abattoir ovaries using fresh, frozen and epididymal spermatozoa from crossbred bulls in the tropics. Oocytes were cultured in maturation media TCM-199 containing 25mM HEPES, 1mM glutamine L, 2.2mg/mL sodium bicarbonate, antibiotics, 22 µg/mL pyruvate, 1µg/mL estradiol-17β, 0.5µg/mL FSH and 0.06 IU hCG and supplemented with 20% heat inactivated estrus cow serum (serum collected in early estrum) at 39oC temperature, 5% CO2 tension with maximum humidity for 24 hours. Oocytes with maximum cumulus expansion were used for in vitro fertilization. Fresh, frozen and epididymal semen from crossbred bulls was used for in vitro fertilization as treatment 1(n =1690), treatment 2(n = 1620) and treatment 3(n = 1710) respectively. Control group of oocytes (1680) were treated in the same protocol along with each treatment group of oocytes in separate in vitro fertilization drop without sperm injection. The in vitro fertilization medium consisted of Fert-TALP medium supplemented with 1µM epinephrine, 10µM hypotaurine, 20µM pencillamine and 0.56µg/ml heparin. Culture conditions set for IVF were 39oC temperature, 5% CO2 tension with maximum humidity. Oocytes showing sperm penetration evidence like presence of enlarged sperm head, male pronuclei with its accompanying sperm tail in the cytoplasm, oocytes with two pronuclei and a clear second polar body but without a sperm tail were considered as fertilized. None of the oocytes in control group showed cleavage due to parthenogenetic activation. Significantly higher results of fertilization rate (p&lt;0.05) were observed when oocytes inseminated with epididymal spermatozoa than fresh ejaculated semen followed by frozen semen.Int J Appl Sci Biotechnol, Vol 4(2): 166-171

168 EFFECT OF CYCLIC ADENOSINE MONOPHOSPHATE MODULATORS DURING OOCYTE IN VITRO MATURATION ON BOVINE EMBRYOS (Gyr×HOLSTEIN) QUALITY

In vitro embryo production (IVP) is an important tool for cattle breeding. Brazilian dairy systems are based on Gyr × Holstein crossbreds, which integrates adaptability to tropical conditions and milk production. Quality determines the oocyte proportion that will develop to blastocyst stage, and although the lipid content is important in oocyte development, a high concentration in embryos is associated with cryotolerance reduction, making this a relevant issue for IVP systems. The in vitro maturation system (IVM) simulated physiological oocyte maturation (SPOM) mimics the physiological maturation events by using cyclic adenosine monophosphate (cAMP) modulators, which promote the increase of oocyte competence. Among the modulators, Forskolin has lipolytic properties. The aim of this study was to evaluate the effect of the SPOM system (Albuz 2010 Hum. Reprod. 25, 12) on bovine embryos (Gyr × Holstein) regarding their total number of cells (TNC) and lipid content. Oocytes were obtained by ovum pick-up from Gyr cows in 5 replications. After selection, they were randomly divided into 2 groups: SPOM (S) and control (C). The IVM lasted 24 h for group C (TCM 199 medium without FBS) in culture oven at 38.5°C, 5% CO2 in atmospheric air and high humidity. In the SPOM system, oocytes were in pre-IVM [TCM 199 medium + 100 µM Forskolin + 500 µM 3-isobutyl-1-methylxanthine (IBMX)] for 2 h and followed for extended IVM (TCM 199 medium + 20 µM cilostamide) for 28 h under the same conditions as control group. After IVM, oocytes were fertilised with semen from a single Holstein bull that was prepared by Percoll gradient method in Fert-TALP medium (Bioklone® Animal Reproduction, São Paulo, Brazil) for 22 h and transfered to culture droplets, where they remained for 7 days (n = 10–13 per group). The lipid content analysis was performed by staining with Oil red and the stained area fraction of each embryo was measured using software ImageJ (NIH, Bethesda, MD, USA). The TNC was measured after being stained with Hoechst 33342 and results were analysed by Student's t-test in Instat GraphPad program, with a 5% significance level. There was no significant difference (P &gt; 0.05) between embryos from both groups on TNC (group S: 88.9 ± 28.0A; group C: 101.6 ± 29.1a) and lipid content (group S: 0.93 ± 12:18A; group C: ±0.15 to 0.96) analysis. Some studies have shown there is a beneficial effect on embryo quality when using this system; however, our results demonstrated that there was no effect on total number of cells using our conditions. Some authors have also demonstrated a reduction in embryo lipid content using Forskolin during in vitro culture. Our results suggest that the time of Forskolin exposure was not enough to ensure lipolytic action on the structures produced from oocytes (Gyr) treated in pre-IVM. It was concluded that the SPOM system had no effect on TNC and lipid content of Gyr/Holstein embryos. Financial support from FAPERJ and CAPES is acknowledged.

157 EFFECT OF MELATONIN ON EMBRYO QUALITY OF BOVINE OOCYTES SUBJECTED TO HEAT SHOCK

The aim of this study was to evaluate the effect of different concentrations of melatonin added to in vitro maturation (IVM) medium of oocytes subjected to heat shock on embryo quality. Immature oocytes aspirated from ovaries obtained from a slaughterhouse were selected and randomly allocated in factorial experiment design (3 × 2). Three concentrations of melatonin (0, 10–6, and 10–4 M; M5250, Sigma, St. Louis, MO, USA) were added to the IVM medium and 2 incubation conditions (conventional: 24 h at 38.5°C and 5% CO2; heat shock: 12 h at 41°C followed by 12 h at 38.5°C and 5% CO2) were tested, resulting in treatments: M1 (0 M; 38.5°C; n = 15), M2 (10–6 M; 38.5°C; n = 15), M3 (10–4 M; 38.5°C; n = 15), M4 (0 M; 41°C; n = 15), M5 (10–6 M; 41°C; n = 15), and M6 (10–4 M; 41°C; n = 15). The IVM was performed in a Nunc plate (144444 – Thermo, Fisher Scientific Inc., Pittsburgh, PA, USA) containing 400 µL of TCM-199 (Invitrogen, Carlsbad, CA, USA) supplemented with 20 µg mL–1 of FSH (Pluset®, Calier Laboratories, Barcelona, Spain) and 10% oestrus cow serum. Oocytes were IVF in FERT-TALP medium for 20–22 h and incubated at 38.5°C and 5% CO2. After IVF, the presumptive zygotes were denuded and cultured in CR2aa medium supplemented with 2.5% FCS (Nutricell, Campinas, Brazil) in an incubator at 38.5°C under 5% CO2, 5% O2, and 90% N2, and saturated humidity for 8 days. Blastocysts with 8 days post-fertilization from different treatments were fixed in 4% paraformaldehyde in PBS for 1 h and analysed by TUNEL assay (Deadend™ Fluorometric TUNEL System, Promega, Madison, WI, USA) to evaluate embryonic quality. Data were analysed by generalised linear models considering the Poisson distribution and using the Proc Genmod of SAS software (version 9.1; SAS Institute Inc., Cary, NC, USA) considering effects of melatonin concentration, incubation conditions, and interaction between the factors. Values shown are the mean ± s.e.m. The interaction between melatonin concentration and incubation conditions was no significant (P &gt; 0.05). The total number of cells was not affected (P &gt; 0.05) by melatonin, but it was decreased (P &lt; 0.05) by heat shock (M1 = 117 ± 6.7a; M2 = 118 ± 4.2a; M3 = 120 ± 6.3a; M4 = 102 ± 6.2b; M5 = 106 ± 5.7b; M6 = 108 ± 8.9b). Melatonin and heat shock did not affect (P &gt; 0.05) the index of embryo apoptotic cells (M1 = 15.3% ± 2.0; M2 = 15.5% ± 1.3; M3 = 13.6% ± 2.0; M4 = 14.9% ± 1.5; M5 = 13.3% ± 1.3; M6 = 13.5% ± 1.2) and the index of trophoblast cells (M1 = 74.6% ± 2.3; M2 = 75.0% ± 1.7; M3 = 75.2% ± 1.9; M4 = 78.4% ± 2.3; M5 = 76.4% ± 3.0; M6 = 75.2% ± 2.6). The melatonin and heat shock affected the index of the inner cell mass (ICM; P &lt; 0.05), and the heat shock reduced the index of the ICM of oocytes not treated with melatonin (M1 = 25.4% ± 2.3a; M2 = 25.0% ± 1.7a; M3 = 24.8% ± 1.8a; M4 = 21.6% ± 2.3b; M5 = 23.6% ± 3.0a; M6 = 24.8% ± 2.6a). In conclusion, melatonin supplementation to the medium IVM of oocytes subjected to heat shock had no effect on blastocyst total cell number, general apoptotic index, or index of the trophoblast cells, but increased index of the ICM. Research was supported by Fapemig, CNPq, Embrapa, and CAPES.

162 EFFECTS OF PROLACTIN AND DITHIOTHREITOL ON THE QUALITY AND DEVELOPMENTAL CAPACITY OF IN VITRO MATURED BOVINE OOCYTES

In vitro maturation (IVM) and IVF of domestic animal oocytes is widely used for commercial and research purposes. The oocyte quality and capacity for further development acquired during in vitro maturation and reduced during the subsequent aging are the main limitative factors affecting the embryo production (Miao et al. 2009 Hum. Reprod. Update 15, 573–585). Our objective was to evaluate effects of prolactin (PRL) and dithiothreitol (DTT) on apoptosis and the embryo development of bovine oocytes matured in vitro using 2 different systems. A total of 1437 slaughterhouse-derived cumulus-oocyte complexes (COC) were matured for 24 h in TCM-199 supplemented with 10% FCS, 0.2 mM sodium pyruvate, 10 μg mL–1 porcine FSH, and 5 μg mL–1 ovine LH. In system 1, 251 COC from a total of IVM oocytes were transferred to the aging medium (TCM-199 supplemented with 10% FCS) and cultured for 24 h in the absence (control) and the presence of either PRL (20 and 50 ng mL–1) or DTT (2.5, 5, and 10 μM). At the end of culture, oocyte apoptosis was detected using the TUNEL method. In system 2, another part of IVM oocytes (1186 COC) was co-incubated for 18 h with sperm in Fert-TALP medium modified by addition of 10 μg mL–1 heparin, PHE (20 μM penicillamine, 10 μM hypotaurine, 1 μM epinephrine), and 0.1% modified Eagle’s medium (MEM) nonessential amino acids. In this case, PRL and DTT (at the above listed concentrations) were added directly to the fertilization medium. After IVF, oocytes were cultured in CR1aa medium for assessment of the cleavage and blastocyst rates on Days 2 and 8, respectively. The nuclear status of blastocysts was evaluated by the cytogenetic method. The data from 3–7 replicates were analysed by ANOVA. Culture of matured COC in the aging medium (system 1) increased the rate of apoptotic oocytes from 8.1 ± 4.7% (0 h) to 48.6 ± 5.8% (24 h) (P &lt; 0.01). This rate was reduced (P &lt; 0.05) up to 22.5 ± 3.1% and 17.8 ± 5.1% in the presence of PRL (20 and 50 ng mL–1) and up to 15.0 ± 6.9% and 19.5 ± 3.7% in the presence of DTT (2.5 and 5 μM). The direct addition of PRL at a concentration of 20 ng mL–1 to the IVF medium raised the blastocyst rate from 21.6 ± 2.2% to 29.8 ± 2.4% (P &lt; 0.05) but did not affect the cleavage rate (72.1 ± 2.2% v. 74.3 ± 2.1%). By contrast, 50 ng mL–1 PRL did not increase the yield of blastocysts and decreased the cleavage rate (from 74.3 ± 2.1% to 62.9 ± 2.4%, P &lt; 0.05). When added to the IVF medium, DTT raised the blastocyst rate only at a concentration of 2.5 μM (P &lt; 0.05). No effects of PRL and DTT on the number of cells in embryos at the blastocyst stage were found. Our findings indicated that PRL and DTT supplements during in vitro fertilization of bovine oocytes may improve their capacity for the subsequent embryo development. This effect was probably due to the inhibitory influence of PRL and DTT on apoptosis of matured oocytes. The study was supported by the Federal Agency for Scientific Organizations and RFBR (project No. 14–48–03681).

Purification of binder of sperm protein 1 (BSP1) and its effects on bovine in vitro embryo development after fertilization with ejaculated and epididymal sperm

The present study evaluated functional aspects of binder of sperm 1 (BSP1) in the bovine species. In a first experiment, cumulus-oocyte complexes (n = 1274) were incubated with frozen-thawed ejaculated sperm (18 hours) in Fert-TALP medium containing: heparin, 10, 20, or 40 μg/mL BSP1. Heparin followed by gelatin affinity chromatography was used for purification of BSP1 from bovine seminal vesicle fluid. With ejaculated sperm, cleavage rates were similar when Fert-TALP medium was incubated with heparin (74.1 ± 2.7%), 10 μg/mL BSP1 (77.8 ± 3.1%), or 20 μg/mL BSP1 (74 ± 2.0%). Day-7 blastocyst rates were equivalent after incubations with heparin (40.8 ± 5.0%) and 10 μg/mL BSP1 (34.1 ± 4.4%), but reduced after 20 μg/mL BSP1 (22.4 ± 2.9%) and 40 μg/mL BSP1 (19.3 ± 4.1%; P < 0.05). In the second experiment, cumulus-oocyte complexes (n = 1213) were incubated with frozen-thawed cauda epididymal sperm (18 hours) in Fert-TALP medium containing: no heparin, heparin, 10, 20, or 40 μg/mL. Cleavage and blastocyst rates were similar after treatments with heparin (68.5 ± 1.3% and 24.7 ± 3.2%, respectively) or without heparin (65.5 ± 1.8% and 27.3 ± 1.6%, respectively). Cleavage was higher after treatment with any BSP1 concentrations (74.2 ± 2.7%–79.0 ± 1.1%) than without heparin (P < 0.05). Also, cleavage was better after Fert-TALP medium incubation with 40 μg/mL BSP1 (79.0 ± 1.1%) than with heparin (68.5 ± 1.3%; P < 0.05). Embryo development was higher (P < 0.05) after treatment with 20 μg/mL BSP1 (35.6 ± 2.5%) and 40 μg/mL (41.1 ± 2%) than after incubations with heparin (24.7 ± 3.2%) or without heparin (27.3 ± 1.6%). Interestingly, BSP1 did not cause reductions in blastocyst rates after fertilization with epididymal sperm, as observed with ejaculated sperm. On the basis of immunocytochemistry, there was BSP1 binding to frozen-thawed ejaculated but not to epididymal sperm. Also, anti-BSP1 reaction remained on ejaculated sperm (as expected) and appeared on epididymal sperm after incubation with purified BSP1. Acrosome reaction of ejaculated and epididymal sperm was induced after incubation with purified BSP1 as well, indicating an effect of BSP1 on capacitation. In conclusion, purified BSP1 from bull seminal vesicles was able to bind to and induce capacitation of ejaculated and epididymal sperm. Also, BSP1 added to fertilization media and allowed proper cleavage and embryo development, with the effects being modulated by previous exposure or not of spermatozoa to seminal plasma.

256 EFFECT OF SPERM SELECTION ON THE RATE OF IN VITRO FERTILIZATION IN ALPACA (VICUGNA PACOS)

In alpacas, improvement of reproductive efficiency of male camelids is limited by the small size of the testes, extended period of ejaculation, and low quality of semen. This study was designed to determine the effect of 2 sperm preparation treatments before IVF on the cleavage rate. The sperm was obtained by slicing the head of the epididymis of slaughtered male alpacas (n = 8), diluting in Tris-yolk-glycerol, and freezing with the slow-cooling method. Frozen semen straws per each male were thawed in a water bath at 37°C for 15 s and evaluated for percentage of progressive motility (32 ± 8.6%) and concentration (66.5 ± 24 × 106 sperm mL–1) post-thawing. Sperm selection by the swim-up method was performed by centrifugation at 1077 × g for 5 min with washing sperm medium eliminating the supernatant; sperm were settled in inclined tube with fertilization medium (without capacitating agent) for 60 min, after which 100 μL from the surface was recovered for use in IVF. The washing method consisted in repeated washing (twice) of sperm in washing sperm medium and fertilization medium by centrifugation at 1077 × g for 5 and 3 min, respectively, and recovery of 50 μL from the bottom of the tube for use in IVF. Sperm selected by swim-up or washing methods had similar characteristics of progressive motility (18 and 23%); however, the concentration was higher for the washing v. swim-up method (52 v. 14 × 106 sperm mL–1, respectively). Cumulus-oocyte complexes (COC) were recovered from 278 ovaries of alpacas killed at abattoirs and classified (Grade 1 and 2) for in vitro maturation (38.5°C at 5% CO2 in air for 27 h in 50 μL of 10 COC per drop). A total of 839 oocytes cultured for 27 h in maturation medium were partially stripped out of cumulus cells by gentle aspiration with a pipette. Sperm suspensions in Fert TALP medium (5 μL) from each treatment group were added to each fertilization drop with 10 oocytes per drop of 45 μL obtaining a final concentration of 10 × 106 sperm mL–1 and cultivated for 72 h until their evaluation. The data for the 13 repetitions of the rate of cleavage (2 to 8 cells) were converted to angular values (angle = arcsin √%) with the object of normalizing the distribution of the data; the analysis of variance was performed (complete randomised design with sub-sampling, P &lt; 0.05) using SAS® version 8.0 for Windows. The rate of cleavage (cell division) did not show statistical differences (P = 0.67) for the swim-up method (37%; 155/421) v. washing method (35%; 147/418). The methods of sperm selection (swim-up and washing) did not affect the rate of IVF.

81 EFFECT OF IN VITRO CULTURE SYSTEM MODIFICATION USING CR1aa MEDIUM ON EMBRYO DEVELOPMENT AND PREGNANCY RATE IN CATTLE

The culture of in vitro matured and fertilized oocytes is a critical step of in vitro production of bovine embryos. Generally, oocytes are co-incubated with sperm in TALP medium containing different additions and then zygotes are transferred to a medium with another composition. At the same time the effect of the medium alteration on the development of early embryos is unknown. Continual adjustment of fertilized oocytes to the changing culture environment may result in a reduction of their developmental potential. The aim of the present study was to compare effects of two different culture systems on the embryo development and subsequent pregnancy rate in cattle. Slaughterhouse-derived cumulus–oocyte complexes were matured for 24 h in TCM-199 supplemented with 10% FCS, 0.2 mM sodium pyruvate, 10 μg mL–1 porcine FSH, and 5 μg mL–1 ovine LH. Frozen/thawed sperm from different Russian Black Pied bulls were prepared in Sperm-TALP medium by swim-up procedure. In vitro matured oocytes were co-incubated for 18 h with prepared sperm in the modified Fert-TALP medium containing 10 μg mL–1 heparin, PHE (20 μM penicillamine, 10 μM hypotaurine, 1 μM epinephrine), and 0.1% MEM nonessential amino acids. The embryo culture was carried out using 2 systems. A total of 340 presumptive zygotes were incubated in CR1aa medium (Rosenkrans et al. 1994 J. Anim. Sci. 72, 434–437) up to Day 5 post-insemination (System 1) and a total of 442 presumptive zygotes were incubated for 24 h in a fresh Fert-TALP medium without PHE and heparin and then cleaved embryos were transferred to CR1aa medium and incubated until Day 5 post-insemination (System 2). Thereupon, the embryos were transferred to a fresh CR1aa medium supplemented with 5% FCS and cultured for 3 or 5 days. The embryo development was evaluated at Days 2, 8, and 10 for cleavage and blastocyst formation and hatching rates, respectively. A portion of blastocysts (of Grade 1 according to IETS classification) obtained at Day 8 were immediately transferred to recipients or were frozen in 1.5 M ethylene glycol and stored in liquid nitrogen until transplantation. The embryo development data (from 6–8 replicates) were analysed by ANOVA and the embryo transplantation data were analysed using the chi-squared test. The cleavage rates did not differ among Systems 1 and 2 and were 63.6–65.7%. On the other hand, the significant differences between culture Systems 1 and 2 were detected in rates of blastocysts (21.9 ± 1.4 v. 28.8 ± 2.8; P &lt; 0.05) and hatched blastocysts (7.2 ± 1.2 v. 12.3 ± 1.6; P &lt; 0.05). The pregnancy rate for frozen embryos was also higher (but not significantly) in System 2 than in System 1 [26.3% (9/34) v. 16.7% (2/12)], whereas for fresh embryos the similar values of the pregnancy rate were observed [on average 42.9% (6/14)]. Thus the additional 24-h culture of zygotes in Fert-TALP medium favourably affects bovine embryo development in vitro.

Effect of somatic cell co-culture on cleavage rate and embryo development of in-vitro fertilized goat oocytes

The present study was undertaken to estimate effect of granulosa cell monolayer and oviductal epithelial cells co- culture on cleavage rate and embryo development of in-vitro matured and fertilized goat oocytes. The oocytes were matured in maturation media (TCM-199). After 27 h, matured oocytes were picked up and denuded and then transferred in fert-TALP medium. The capacitated sperm having concentration of 1×106 live sperm/ml were used for in-vitro fertilization. Oocytes were co-incubated with capacitated spermatozoa for 18 h at 38.5±1°C in an atmosphere of 5% CO2 in humidified air. The fertilized oocytes culture in EDM over their respective cell culture i.e. granulosa cells monolayer and oviductal epithelial cells were obtained for further cleavage rate and embryo development (2, 4, 8, and 8 celled) under steriozoom microscope at every 24 h with 50% replacement of pre-equilibrated EDM media up to 7 days. The cleavage rates with granulosa cells monolayer and oviductal epithelial cells co-culture, 48 h post insemination, were 43.52 and 45.27% respectively for goat oocytes. The present study indicated that both granulosa cell monolayer and oviductal epithelial cells co-culture behaved similarly for cleavage rate of in-vitro matured and fertilized goat oocytes, and oviductal epithelial cells co-culture significantly increased in embryonic development stage from 4 cell to 8 cell stage (4 cell, 8 cell and &gt; 8 cell) as compared to granulosa cell monolayer whereas granulosa cell monolayer co- culture significantly increased the embryonic development of 2 cell stage as compared to oviductal epithelial cells.