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The conservation of female animal genetics is challenging because of the scarcity of oocytes and their sensitivity to cryopreservation techniques. During slow, controlled freezing procedures, intracellular ice crystallization often leads to cell damage. Vitrification as an alternate method of cryopreservation exposes cells to a higher concentration of cryoprotectants with an ultra-rapid cooling rate, leading them to an ice-crystal-free, solid glasslike structure. The vitrification procedure has been used successfully for the cryopreservation of embryos and other body tissues, but very few reports of successful oocyte cryopreservation exist because of their complex structure. The present study was designed to compare two packaging methods (Cryotop v. 0.25-mL straw) and two equilibration times (10 v. 0 min) for vitrification of bovine oocytes. COC were aspirated from follicles 0.05) in either packaging method. In conclusion, vitrification of bovine oocytes using the Cryotop method provides an alternative for the cryopreservation of bovine oocytes. Moreover, bovine oocytes can be successfully vitrified without equilibration.This study was supported by the Canadian Animal Genetic Resources Program, Agriculture and Agri-Food Canada. [ABSTRACT FROM AUTHOR]

Successful cryopreservation of embryos depends on the pre-equilibration time. This study was designed to compare 2 pre-equilibration times ? short (1 min) and long (5 min) ? and to evaluate the re-expansion and hatching rates of different stages of embryos using the short pre-equilibration method. Cumulus?oocyte complexes (COCs) from ovaries obtained from a slaughterhouse were matured, fertilized, and cultured in vitro. In Experiment 1, expanded blastocysts between 7 and 9 days of culture were pre-equilibrated for the short time (1 min) in 100 ?L of vitrification solution 1 (VS1: containing 7.5% ethylene glycol (EG), 7.5% dimethyl sulfoxide (DMSO), and 20% calf serum (CS) in TCM-199), followed by incubation in 100 ?L of vitrification solution 2 (VS2: containing 15% EG, 15% DMSO, 20% CS, and 1 m sucrose (Suc) in TCM-199) for 30 s. Another group of blastocysts was subjected to the long pre-equilibration (5 min) in 100 ?L VS1, followed by incubation in 100 ?L of VS2 for 30 s. The embryos were placed into Cryotops (Kitasato Supply Co., Tokyo, Japan) and immediately submerged in liquid nitrogen and kept there for 1 week. Blastocysts were warmed by plunging the Cryotops into 1 m Suc in TCM-199 for 1 min, placed in 0.5 m Suc in TCM-199 for 3 min, and finally placed in CR1aa alone for 5 min before being cultured. In Experiment 2, 8-cell embryos, morulae, and expanded blastocysts were vitrified by the previously described short equilibration method. The re-expansion and hatching rates of embryos were determined as the percentage of vitrified?warmed embryos undergoing further development in the in vitro culture. The data were analyzed by the chi-square test. Results are presented in Table 1. There was no difference between the short and long pre-equilibration times in terms of survival (94.0% v. 94.1%, respectively) and morphological appearance immediately after warming. However, re-expansion of blastocysts (ability to develop further) was slightly higher with the short pre-equilibration than with the long pre-equilibration (90.0% v. 85.9%, respectively). In Experiment 2, there were no differences in embryo re-expansion, but the hatching rates of 8-cell embryos were lower than those of morulae, blastocysts, and controls. In conclusion, the results of this study indicate that the length of pre-equilibration time prior to vitrification does not influence embryo re-expansion, and that bovine morulae and blastocysts can be vitrified with equal success. We also conclude that insufficient permeation of cryoprotectants may occur in 8-cell embryos. [ABSTRACT FROM AUTHOR]

The objective of this study was to find an optimal parameter (liquid-ice interface velocity; velocity) of the directional freezing method (Arav et al. 2002 Reprod. Nutr. Dev. 42, 583–586) for conventionally processed bull sperm stored in 0.5-mL straws. We also evaluated sex-sorted bull sperm frozen using this method. Experiment 1: Each single ejaculate from two Holstein bulls was split and processed in egg yolk citrate extender (2 steps, 6% glycerol final; EYC) or egg yolk Tris extender (1 step, 7% glycerol final; EYT1). After cooling and loading in 0.5-mL plastic straws, each batch of semen was frozen using a directional 0.5-mL straw freezer (MTG 550; IMT, Ltd., Ness-Ziona, Israel) in 6 different velocities (v2.0, v2.2, v2.4, v2.6, v2.8, v3.0; mm s-1). Ice seeding time was 45 s. Static freezing in liquid nitrogen vapor was used as the control. Sperm parameters were measured using computer-assisted semen analysis (CASA) at 0, 1, and 2 h after thawing. The thawed samples were also evaluated for sperm viability and acrosomal integrity by triple staining. The experiment was repeated 3 times. Data were analyzed by ANOVA. No significant differences in general and progressive motility were observed in each extender regardless of the freezing method. However, sperm viability and acrosomal integrity of sperm frozen in EYC were highest at v3.0 (73.2% and 84.7%, respectively) and were generally higher after MTG freezing (63.4 to 73.2% and 80.6 to 84.7%, respectively) than in the control (52.6% and 73.1%, respectively; P < 0.05 or 0.01) except for v2.4 (54.5% and 70.8%). Sperm viability (74.6 to 77.9% vs. 63.2%) and acrosomal integrity (84.8 to 88.9% vs. 79.3%) in EYT after MTG freezing were also higher than in the control (P < 0.01). Experiment 2: Two single ejaculates from a Holstein bull were flow cytometrically sex-sorted (Schenk et al. 1999 Theriogenology 52, 1375–1391) for X sperm. Each ejaculate was assigned to processing in EYC or egg yolk Tris extender (2 steps, 6% glycerol final; EYT2). Processed sperm was frozen using MTG 550 (velocity set at v3.0) or in liquid nitrogen vapor as control and analyzed as in Experiment 1. In the control, general (40.2% vs. 21.1%; P < 0.01) and progressive (16.3% vs. 4.3%; P < 0.05) motility in EYC were higher than in EYT2, respectively, at 0 h. In MTG freezing, motility (at 0 h, 42.0% vs. 28.3%; P < 0.05), viability (60.5% vs. 40.8%; P < 0.01), and acrosomal integrity (86.9% vs. 71.0%; P < 0.05) in EYC were higher than in EYT2, respectively. But in experiment 2, there were no differences in motility, progressive motility, viability, and acrosomal integrity between MTG freezing and control. The above results suggest that MTG freezing improves membrane and acrosomal quality of bull sperm frozen in 0.5-mL straws. Faster interface velocity (3.0 mm s-1) seems optimal for the given condition. Egg yolk citrate extender seems to be beneficial for MTG freezing of sex-sorted bull sperm, but further studies using ejaculates from different bulls are required to confirm the apparent benefit. [ABSTRACT FROM AUTHOR]

Ultrasoundsonography (US) is an essential tool for the study of reproductive physiology and is particularly useful in research on ovarian follicular dynamics in cattle. The resolution of the US images obtained, however, differs according to the frequency of the transducer used. The objective of this study was to investigate the effect of transducer frequency on the accuracy of follicle identification in cattle. A Honda HS-2000 sonograph equipped with 5.0, 7.5, and 10.0 MHz of B-mode linear rectal transducers was used in this study. A total of 22 ovaries with corpus luteum were collected from a local slaughterhouse. Each ovary was fixed on a paraffin block, immersed in de-gassed water, the transducer was placed at a distance of 1 cm from the surface, and sequential images from one end to other end of ovary were obtained at a distance of 1 mm. All follicles were aspirated by needle after imaging and were then injected with a pigment after counting by all frequencies. Subsequently, following freezing, 5-mm sections of ovaries were cut and the number of follicles in the sections was counted using a megascope. The follicles were classified into 4 groups according to their diameter: 2 to 3 mm, 4 to 6 mm, 7 to 10 mm, and 11 mm. The number of follicles observed using the megascope was compared with those observed at the 3 US frequencies. The ovaries were classified according to corpus luteum (CL) by determining the condition of the CL. Very red and small-sized CL (stage 1: ovary ovulated within 3 days), elastic and bigger CL (stage 2: ovary ovulated at 4 to 14 days), and regressed CL with yellow color (stage 3: ovary ovulated at 15 to 20 days). The data were analyzed by ANOVA. The results revealed no difference (P> 0.05) in the number of follicles observed in any size category using the US transducers and the megascope. Further, there were no differences between the different transducer frequencies and megascopic observations in terms of the number of follicles ≥4 mm in diameter. The stage of estrus in the ovary also had no effect on the number of follicles observed regardless of follicular size or the US frequency used. In conclusion, these results indicated that follicles could be counted accurately in excised ovaries using 5.0-, 7.5-, and 10.0-MHz transducers.Table 1.The average number of follicles in different frequencies and megascopic (n= 22) [ABSTRACT FROM AUTHOR]

Prolonged exposure of oocytes to cryoprotectants causes cell injury, whereas a short exposure time results in insufficient permeation because of ice formation. This study was designed to determine the optimal pre-equilibration time and its effect on the survival rate of matured bovine oocytes after warming and on subsequent embryo development. Bovine COC were matured for 20 h in TCM-199 supplemented with 5% calf serum (CS) and 0.02 AU mL?1 of FSH at 38.5C in 5% CO2 in air. Then, the COC were partially denuded. The oocytes were pre-equilibrated in 100 ?L of vitrification solution 1 (VS1) containing 7.5% ethylene glycol (EG), 7.5% DMSO, and 20% CS in TCM-199 for 0, 1, 3, and 5 min. Then, the oocytes were moved through 100-?L drops of vitrification solution 2 (VS2) containing 30% EG, 30% DMSO, 0.5 m sucrose (Suc), and 20% CS in TCM-199 for 30 s, loaded into cryotops, and immersed into liquid nitrogen. Oocytes were warmed by plunging the cryotops into 1 m Suc in TCM-199 supplemented with 20% CS for 1 min, placed in 0.5 m Suc in TCM-199 supplemented with 20% CS for 3 min, and finally in TCM-199 supplemented with 20% CS alone for 5 min. Frozen?thawed semen from a single bull (5 106 spermatozoa mL?1) was used for fertilization. Zygotes were vortexed to remove the cumulus cells 18 h after fertilization and cultured in CR1aa for 9 days. Data were analyzed by the chi-square test. Results are presented in Table 1. There were no differences in the survival rates of the control and vitrified oocytes. The cleavage rate of controls at both 24 and 48 h was greater (P < 0.01) than that of vitrified oocytes. Among pre-equilibration times, the cleavage rate of 0, 1, and 3 min pre-equilibrations at both 24 and 48 h was greater than with the pre-equilibration time of 5 min (P < 0.01). With respect to blastocyst development, the control oocytes showed greater development rates than the vitrified oocytes (P < 0.01), whereas the development rates were lower with the pre-equilibration time of 5 min (P < 0.01) than with the other pre-equilibration times. In conclusion, the results indicated that matured bovine oocytes could survive after vitrification and subsequently develop into blastocysts after IVF. However, the pre-equilibration time before vitrification affects cleavage and blastocyst development; a longer exposure time resulted in lower blastocyst development. [ABSTRACT FROM AUTHOR]

The objective was to investigate the use of Cue-Mate and porcine LH (pLH) or GnRH with or without presynchronization on pregnancy rate following timed AI (TAI) in Angus heifers (n = 462). Approximately half of the heifers (Control; n = 236) were treated at random stages of the estrous cycle, and the other half (Presynch; n = 226) received two injections of 500 g cloprostenol (PGF: Estrumate; Schering Plough Animal Health, Pointe-Claire, Quebec, Canada) 14 days apart; synchronization treatments were initiated 11 days after the second injection of PGF. On Day 0, heifers received an intravaginal progesterone device (Cue-Mate; Bioniche Animal Health, A/Asia Pty, Armidale, Australia) and were treated with 100 g GnRH IM (Cystorelin; Merial Canada Inc., Victoriaville, Quebec, Canada; n = 233) or 12.5 mg pLH (Lutropin-V; Bioniche; n = 229). On Day 7, Cue-Mates were removed and heifers were given PGF IM. GnRH or pLH (same as the first treatment) was given concurrently with TAI on Day 9 (54–56 h after PGF) with frozen–thawed semen of one of 3 sires. Ultrasonographic examinations were performed in a subset of 182 heifers on Days 0 and 7 for CL and follicle development, and in all heifers on Days 41 to 49 for confirmation of pregnancy. Data were compared using CATMOD procedures in the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA). The proportion of cycling heifers on Day 0 was 90.7% (165/182). Pregnancy rate tended to differ among bulls (46.7, 56.9, and 61.4% for Bulls A, B, and C, respectively; P? 0.1). Heifers that ovulated in response to the first GnRH or pLH injection had a higher pregnancy rate than those that did not ovulate (66.3 vs. 51.9%; P ? 0.03). In addition, heifers treated with GnRH tended to have a higher ovulatory response to the first treatment and a higher pregnancy rate to TAI than those treated with pLH (60.9 and 60.5% vs. 50.0 and 52.4%, respectively; P ? 0.09). Although ovulatory response to the first GnRH or pLH treatment was 46.5 and 63.5% for Control and Presynch groups, respectively (P ? 0.02), pregnancy rates did not differ (59.8 vs. 53.1%; P ? 0.2). However, there was an interaction between presynchronization treatment and ovulatory response to the first injection of GnRH or pLH on pregnancy rates (P ? 0.02). The pregnancy rate was higher in Control heifers that ovulated (77.5%) to the first injection of GnRH or pLH than in Control heifers that did not ovulate (52.2%) or Presynch heifers that did (59.0%) or did not (51.4%) ovulate. In summary, Cue-Mate-treated heifers that ovulated in response to the first GnRH or pLH treatment had higher pregnancy rates to TAI. Although presynchronization with PGF increased the ovulation rate, it did not significantly affect the pregnancy rate. [ABSTRACT FROM AUTHOR]

The morphological identification of immature oocytes has commonly been used to select the bovine oocytes for IVF. However, <30? of the recovered oocytes reach the blastocyst stage after fertilization, and this is probably due to the quality of the oocytes at the beginning of maturation. The brilliant cresyl blue (BCB) stain determines the activity of glucose-6-phosphate dehydrogenase, an enzyme synthesized in growing oocytes. The aim of this study was to evaluate the effect of the BCB stain on the selection of bovine oocytes and on the subsequent embryo development for in vitro production (IVP). Cumulus?oocyte complexes (COCs) were collected by the aspiration of 2- to 6-mm follicles. A total of 559 oocytes were divided into 2 groups: (1) a control group, immediately cultured, and (2) a BCB-incubated group. After 90 min of BCB staining (Pujol et al. 2004 Theriogenology 61, 735?744), the oocytes were divided into oocytes with blue cytoplasm (BCB?) and oocytes without blue cytoplasm (BCB-). The COCs were matured for 20 h in TCM-199 supplemented with 5? calf serum (CS) and 0.02 mg mL-1 FSH at 38.5C under an atmosphere of 5? CO2 in air. The matured COCs were inseminated with 5 106 sperm mL-1. After 18 h of gamete co-culture, the presumed zygotes were cultured in CR1aa supplemented with 5? CS for 9 days at 38.5C under an atmosphere of 5? CO2, 5? O2, and 90? N2. Embryonic development was evaluated at 48 h after IVF (proportion of ?5-cell stage, the total cleavage rates) and on Days 7 to 9 (blastocyst rate). The experiment was replicated 5 times, and the data were analyzed by a chi-square test and ANOVA. The results are presented in Table 1. The proportion of embryos with ?5-cell stage was significantly higher (P < 0.01) in the BCB? group than in the BCB- group, but not in the control group. The total cleavage rate for the BCB? embryos was significantly higher than that of either the BCB- or the control group (P < 0.01). There were also significant differences (P < 0.01) in the blastocyst development between the BCB? and BCB- embryos and between the BCB- and the control embryos (P < 0.05). This result showed that the selection of bovine oocytes by BCB staining before in vitro maturation may be useful for selecting oocytes that are developmentally competent up to Day 9 for IVP. [ABSTRACT FROM AUTHOR]

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.

Embryo transfer entails many procedures and techniques, of which embryo freezing is an important component in bovine embryo transfer. Embryo freezing techniques have been developed over the last 40 years, allowing practical availability, and have become essential for cattle reproduction management under field conditions. The direct transfer methods of frozen-thawed, in vivo-derived, and in vitro-produced (IVF) bovine embryos using 1.5 M ethylene glycol (EG) with or without sucrose (SUC) are used widely under on-farm conditions, not only in Japan but also globally. The direct transfer method using 1.5 M glycerol (GLY) and 0.25 M SUC (GLY-SUC) is used mainly in Japan. The pregnancy rate with direct transfer of frozen-thawed bovine embryos in either EG or GLY-SUC has been found to not differ from conventional freezing with GLY and traditional dilution techniques. Pregnancy rates following direct transfer of frozen-thawed bovine embryos were affected by the developmental stage of the embryos and the parity of the recipients. The use of ultrasound-guided on-farm ovum pickup is ushering in a new revolution for the commercial application of IVF embryos. Globally, for the first time more IVF bovine embryos were transferred in 2017 than produced in vivo. More than 60% of IVF embryos were transferred fresh due to a low pregnancy rate of frozen-thawed IVF embryos. Many factors seemed to be involved in improving the survival rate of frozen-thawed IVF embryos. Therefore, further research is needed to improve the freezing tolerance of IVF embryos to develop efficient direct transfer methods analogous to those used for in vivo embryos.

Generating techniques to enhance the success of blastomere separation is important for bovine economy, because it increases the number of transferable embryos. This study aimed to identify the optimum cryoprotectants for the vitrification of bovine embryos and the separation of blastomeres at different stages. In experiment 1, expanded blastocysts were vitrified in two different vitrification solutions, either (1) ethylene glycol (EG) + propylene glycol (PG) or (2) EG. The survival rate of blastocysts in the EG + PG was higher than that of the EG. In experiment 2, intact two-cell and eight-cell stage embryos were vitrified in the same solutions used in experiment 1. The EG + PG produced more dead embryos than the EG (P < 0.05). In the EG, the rate of blastocyst formation was similar for the vitrified two- and eight-cell embryos and the non-vitrified ywo-cell embryos. In experiment 3, separated blastomeres of two- and eight-cell embryos were vitrified in EG. There was no difference in the rate of blastocyst formation and total number of cells between the two vitrified groups. In summary, at the blastocyst stage, EG + PG was superior, based on both survival rates and cell numbers; however, at the 2-8 cell stage, the use of EG alone was better than the other groups., (© 2017 Japanese Society of Animal Science.)

Recently, gene-editing using the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) technique has attempted to utilize fibroblasts of livestock animals for somatic cell nuclear transfer. In this study, we establish the procedure for preparing skin fibroblast clones whose genes were edited by the CRISPR/Cas9 technique. After isolating fibroblasts from earlobes of Japanese Black cattle, subsequent collagenase-digestion and extensive wash procedures enabled us to avoid contamination of fungi. Electroporation using NEPA21, rather than lipofection using commercially available liposome reagents, allowed us to perform more efficient transfection of plasmid constructs. Although bovine ear-derived fibroblasts were not able to proliferate in single cell cultures in Dulbecco's modified Eagle medium containing 10% fetal calf serum, supplementation with insulin-transferrin-selenium mixture, human recombinant epidermal growth factor, or human recombinant basic fibroblast growth factor promoted proliferation of the cells, even in a single cell culture. Taking advantage of our established protocol, we eventually obtained eight ear-derived fibroblast clones with a recessive mutation in the isoleucyl-tRNA synthetase gene corrected by the CRISPR/Cas9 technique.

This study investigated re-expansion dynamics during culture of bovine blastocysts cryopreserved either by slow-freezing or vitrification. Also, the extent and localization of membrane damage and DNA fragmentation in re-expanded embryos were studied. Frozen-thawed embryos showed a significantly lower re-expansion rate during 24 h of post-thawing culture compared to vitrified embryos. Vitrified embryos reached the maximum level of re-expansion rate by 12 h of culture whereas frozen embryos showed a gradual increase in re-expansion rate by 24 h of culture. When assayed by Hoechst/propidium iodide staining there was no difference in the numbers and ratio of membrane damaged cells between re-expanded frozen and vitrified embryos; however, the extent of membrane damage in blastomeres was significantly higher in both groups compared with non-cryopreserved embryos (control). TUNEL assay combined with differential ICM and TE staining revealed a significantly higher number and ratio of TE cells showing DNA-fragmentation in frozen-thawed re-expanded blastocysts compared to vitrified ones; however, vitrification also resulted in an increased extent of DNA fragmentation in TE cells compared with control blastocysts. In frozen-thawed blastocysts increased extent of DNA fragmentation was associated with reduced numbers and proportion of TE cells compared with vitrified and control embryos. The number and ratio of ICM cells and the extent of DNA fragmentation in ICM did not differ among control, frozen and vitrified groups. In conclusion, compared with vitrified embryos, blastocysts preserved by slow-freezing showed a delayed timing of re-expansion which was associated with an increased frequency of DNA fragmentation in TE cells., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The objective of the study was to determine whether the serum obtained from animals differing in body condition score (BCS) affects in vitro embryo development. After in vitro fertilization, serum obtained from dairy cows of either low (L-BCS; 2.1 ± 0.14 on a scale of 1 to 5) or high BCS (H-BCS; 4.0 ± 0.0), or commercially available bovine serum (control) was added at 5% concentration to the in vitro culture medium. Use of serum obtained from H-BCS cows increased the cleavage rates compared with control serum at both 24 and 48 h after in vitro fertilization (78.3 vs. 71.9% and 79.9 vs. 75.1%, respectively), whereas use of serum obtained from L-BCS cows increased the blastocyst rate compared with control serum at 7d (23.8 vs. 19.1%), but this difference was not evident at 8 or 9 d after in vitro fertilization. As nonesterified fatty acid concentrations were highest in control serum, followed by serum from L-BCS and H-BCS cows (621, 559, and 272 μEq/L, respectively), a high concentration of nonesterified fatty acids might adversely affect the very early stages of embryo development, and its negative effects might be greater immediately after fertilization compared with developmental stages after morula formation. Our findings also indicate that factors promoting early stage embryo development do not necessarily promote blastocyst development. Serum obtained from animals under different physiological conditions may be used for in vitro embryo culture to study the effects of nutritional management of dairy cattle on embryo development., (Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

In the present study, we tested the hypotheses that oocyte competence is compromised by a longer duration of follicular growth and that it is not affected by FSH starvation. Cows were allocated to short FSH (n=14), FSH starvation (n=13) and long FSH (n=13) groups. The first two groups were given eight doses of FSH, whereas the third group was given 14 doses of FSH, starting from the day of wave emergence (Day 0). A progesterone-releasing device (controlled internal drug release; CIDR) was placed intravaginally at the start of the experiment in all groups. The short FSH group was given prostaglandin (PG) F2α on Day 3, whereas the two other groups received PGF2α on Day 6. In all cows, the CIDR was removed at the time of PGF treatment; porcine (p) LH was given 24h after CIDR removal and cows were inseminated 24 and 36 h later. Reproductive tracts were collected 4 days after insemination and ova and/or embryos were cultured for ≥6 days. The FSH starvation group had fewer ovulations (P=0.001), and ova and/or embryos (P<0.05). No difference in embryo quality was detected between long and short FSH groups at 7, 9 or 10 days after artificial insemination. In conclusion, oocyte competence was not altered by the duration of the follicular growth phase in superstimulated cows, whereas FSH starvation substantially reduced the ability of superstimulated follicles to ovulate.

The objective was to investigate the effects of cryodevice, vitrification solutions, and equilibration time on in vitro maturation, cleavage, and embryo development of vitrified bovine oocytes. In Experiment 1, the nuclear maturation (MII) rate of immature bovine COCs vitrified was compared between two equilibration times (0 vs 10 min) in vitrification solution 1 (VS1) and two cryodevices (cryotop vs 0.25 mL straw). The MII rate was higher in the non-vitrified control group than in vitrified groups (61 vs 16%, P < 0.0001). Equilibration time did not affect MII rate (P = 0.964); however, the MII rate was higher for COCs vitrified on cryotops than in straws (23 vs 9%, P = 0.007). In Experiment 2, bovine COCs were vitrified on cryotops using two equilibration times (0 vs 5 min) in VS1 and two kinds of vitrification solutions (freshly prepared vs frozen). Cleavage and blastocyst rates were higher (P < 0.0001) in the non-vitrified control group than vitrified groups (cleavage rate 93 vs 42% and blastocysts rate 31 vs 0.4%). Cleavage rate of COCs vitrified using frozen solutions with 5 min equilibration was higher (P = 0.05) than other treatment groups. However, blastocyst rate did not differ (P = 0.993) among treatment groups. In conclusion, cryotop was a better cryodevice than 0.25 mL straw for vitrification of bovine COCs. Furthermore, 5 min equilibration in VS1 improved cleavage. Compared with control, the vitrification procedure per se damaged bovine COCs, resulting in poor nuclear maturation and embryo development. However, vitrification did not immediately kill oocytes, as the cleavage rate was acceptable., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Although the number of dairy farms is decreasing, that of large farms is increasing in Japan. Milk production in Japanese dairy cows has increased from 62 kg/year to 88 kg/year over the last 2 decades. However, Japanese dairy cows are experiencing a sustained decline in reproductive performance, calving intervals, and days open; further, the number of inseminations required for conception have increased, and the conception rate has decreased. In order to improve fertility in high milk-producing dairy cows, it is necessary to evaluate their reproductive characteristics. In this study, the postpartum body condition score (BCS) was remarkably low, and the functional recovery of reproduction was consequently delayed. Moreover, the results indicate that the estrus duration varies among individual cows. However, it is possible to improve the conception rate by inseminating cows 8-12 h after the onset of estrus. Reproductive management systems suitable for the current dairy farming system with large herd sizes are required.

Repeat breeding is an important factor affecting economic success in dairy management. The objective of this study was to investigate the effectiveness of transfer of frozen-thawed IVF embryos in establishing pregnancy in repeat-breeding Holstein cattle. Cumulus oocyte complexes were collected by aspiration of 2-5 mm follicles from ovaries obtained at two local abattoirs. After IVF, days 7 and 8 blastocysts were frozen either in 1.5M ethylene glycol with 0.1M sucrose, or in 1.4M glycerol with 0.1M sucrose. Holstein recipients (122 heifers and 410 cows) included those that had not conceived after 3-21 inseminations. Embryos frozen in ethylene glycol were transferred directly, and embryos frozen in glycerol were transferred after dilution of the cryoprotectant in sucrose into recipients 7 or 8 days after estrus (without-AI group), or following AI (with-AI group). Pregnancy rates were compared by the Chi-square test. Significantly higher pregnancy rates were achieved by embryo transfer following AI (with-AI group) than by embryo transfer alone (without-AI group) in both heifers (49.2 and 29.5%, respectively) and cows (41.5 and 20.4%, respectively; P<0.05). Pregnancy rates were not significantly different between heifers and cows. However, pregnancy rate decreased as the number of inseminations prior to embryo transfer increased in the with-AI group, but not in the without-AI group. Therefore, transfer of frozen-thawed IVF embryos during the same cycle in which AI was done improved pregnancy rates in repeat-breeding Holstein heifers and cows, and suggested that embryo transfer is an alternative in the treatment of repeat breeding.

Leukemia inhibitory factor (LIF) and macrophage colony stimulating factor (M-CSF), members of the group of hemopoietic cytokines, play a primary role in the control of embryo development and implantation and in the growth of the placenta in humans and mice. Gene expressions of LIF and M-CSF were investigated using quantitative RT-PCR in bovine endometrial tissues during early and mid-pregnancy (Days 16-17, 20-21, 30-36, 48-49 and 74-140) and during the estrous cycle (Days 13-14). Leukemia inhibitory factor and M-CSF genes were expressed in all samples examined. Significant differences were found between the gene expression patterns of LIF and M-CSF. Leukemia inhibitory factor expression level at Days 48-49 was the highest in caruncular endometrium, however, the large variability negated any significant differences. Leukemia inhibitory factor expression levels in intercaruncular endometrium at Days 48-49 and 74-140 of pregnancy were greater than at Days 13-14 of the estrous cycle and at other days of pregnancy. No significant change was recognized in M-CSF expression levels in caruncular endometrium. Macrophage colony stimulating factor expression level in intercaruncular endometrium at Days 74-140 was greater than those of the other samples. These results suggest that LIF and M-CSF are produced in the endometrium and may play different roles in early and mid-pregnancy.

The present study was conducted to examine the effects of culture systems and culture media on developmental competence and freezability of bovine embryos obtained by in vitro culture of in vitro matured and fertilized (IVM-IVF) oocytes. No significant difference was observed in the proportions of oocytes developed to blastocysts, the speed at which the oocytes reached the blastocyst stage and the number of cells, when the IVM-IVF oocytes were cultured in CR1aa with or without cumulus cells. Nevertheless, more of the IVM-IVF oocytes cultured either with or without cumulus cells in CR1aa were seen to reach the blastocyst stage much sooner than those cultured with cumulus cells in TCM199 (P<0.05). The proportion of embryos developed to the blastocyst stage by day 7 in CR1aa culture was significantly higher than embryos cultured in TCM199. Viability after frozen-thawed blastocysts were obtained in vitro, was seen in a significantly higher percentage of embryos cultured in TCM199 and developed to the hatched blastocysts than in those cultured in CR1aa (P<0.05). These results indicate that CR1aa was superior to TCM199 for the potential developmental of IVM-IVF oocytes to blastocysts during in vitro culture regardless of co-culture with or without cumulus cells. But the freezability of blastocysts developed in CR1aa was inferior to those developed in TCM199.

An integrated bovine embryo transfer program was conducted in collaboration with 11 Japanese prefectural livestock experiment stations. The program was conducted to evaluate the practicability of the direct transfer method for bovine embryos frozen-thawed in the presence of propylene glycol (PG) or ethylene glycol (EG) under on-farm conditions. Embryos at the compacted morula to expanded blastocyst stages were collected from superovulated donors on Day 7 or 8 after estrus and equilibrated in 1.6 M PG or 1.8 M EG in Dulbecco's phosphate-buffered saline (DPBS) supplemented with 20% heat-inactivated calf serum. Embryos were then loaded individually into a 0.25-ml straw and placed directly into a cooling chamber of a programmable freezer precooled to -7 degrees C. After 2 min, the straw was seeded, maintained at -7 degrees C for 8 min more, and then cooled to -30 degrees C either at 0.3 degree C/min or 0.5 degree C/min before being plunged into liquid nitrogen. Embryos at the same stages were also frozen in the presence of 1.4 M glycerol (GLY) by a conventional method, which served as a control. The frozen embryos were thawed by allowing the straws to stand in air for 5 to 10 sec and then immersing them in a 30 degrees C water bath. Embryos frozen-thawed in the presence of PG or EG were nonsurgically transferred into the uterine horn without diluting the cryoprotectant. Embryos frozen-thawed in the presence of GLY were nonsurgically transferred after removing GLY either by the stepwise method (GLY-I) or by in situ dilution with 0.3 M sucrose solution (GLY-II). A total of 1,273 (PG: 400, EG: 418, GLY-I: 177, GLY-II; 278) frozen-thawed embryos was transferred into recipients, yielding 545 pregnancies (overall: 42.8%, PG: 36.0%, EG; 44.7%, GLY-I; 48.6%, GLY-II; 46.0%). The pregnancy rate with PG was significantly lower than that with EG or GLY-II (P < 0.05). The pregnancy rate was affected by the type of cryoprotectant, the region where the embryo transfer program was carried out, the developmental stage of the embryos, the parity of the recipients, and corpus luteum (CL) quality of the recipients. There were no differences in rates of abortion and stillbirth among the 3 cryoprotectants. The present study demonstrates that EG can be effectively used as a cryoprotectant for freezing and direct transfer of bovine embryos, and that the direct transfer method is applicable under on-farm conditions.

A sandwich enzymeimmunoassay (EIA) for pregnant mare serum gonadotropin (PMSG) using a microtiter plate was developed. Sensitivity of the assay to PMSG was 15.6 mIU/ml (0.2 ng/well). The PMSG levels in serum were measured with the EIA in superovulated and anti-PMSG rabbit antiserum treated mice and heifers. In mice, the PMSG blood level was measurable in the serum 4-6 days after intraperitoneal injection of 5-30 IU of PMSG. The administration of anti-PMSG antiserum at the same dose level as PMSG caused a rapid decrease in the PMSG blood level, declining to undetectable levels within 17 hours. In heifers, the PMSG level was measurable at 10-11 days after the injection of 2500 or 3000 IU of PMSG. When antiserum was injected 48 hours after the PMSG injection, the clearance rate of PMSG was affected by the route of the administration. The administration of 3000 units of anti-PMSG antiserum intravenously caused a rapid decline and the disappearance of circulating PMSG within 17 hours. When 3000 units of anti-PMSG antiserum was injected intra-muscularly, the PMSG blood level also decreased and became unmeasurable 24 hours after administration; however, it was still detectable for up to 17 hours. These results indicate that the administration of anti-PMSG antiserum at the proper timing and dosage could lead to successful superovulation through the improvement of hormonal conditions.