Birth weight and birth rate of heavy calves
conceived by transfer of in vitro or in
vivo produced bovine embryos
T. Numabe
a,∗, T. Oikawa
a, T. Kikuchi
a, T. Horiuchi
b aMiyagi Prefectural Animal Industry Experiment Station, Miyagi 989-6445, JapanbHiroshima Prefectural University, Shobara, Hiroshima 727-0023, Japan
Received 17 February 2000; received in revised form 30 June 2000; accepted 18 July 2000
Abstract
The aim of this study was to evaluate the difference in birth weight and gestation length between Japanese Black calves obtained from transfer of bovine embryos produced in vitro (IVP) and those developed in vivo (IVD). An additional objective was to clarify the sire effect on birth weight and gestation length and to examine the birth rate of heavier calves. Two Japanese Black bulls breed at our experimental station were used as a semen source for production of IVP and IVD embryos. Thirty-eight Japanese Black heifers and cows of various genetic backgrounds were used as embryo donors for IVD embryos. Ovaries for IVP embryos were collected at random at a local slaughterhouse from Japanese Black cattle of various genetic backgrounds. IVP embryos were
produced using co-culturing with cumulus cells in 5% CS+TCM 199. Both the IVD and IVP
embryos were transferred non-surgically to Holstein recipients on day 7±1 of estrous cycle. In this
study, the birth weights and gestation lengths of half-sib single calves for bull A and B were analyzed. The numbers of single calves born by transfer of IVP and IVD embryos for bull A and B were 133 and 121, 243 and 465, respectively. The birth weight of the IVP calves was significantly higher (P <0.01) than that of the IVD (bull A: 31.0±0.4 kg versus 27.2±0.4 kg and bull B: 29.9±0.6 kg versus 26.6±0.2 kg). Gestation length of the IVP calves for bull A was significantly longer (P <0.01) than that of the IVD (291.9±0.9 days versus 283.6±0.5 days). However, for bull
B, there were no differences in gestation length between the IVP and IVD calves (285.9±0.7 days
versus 286.2±0.3 days). These results clearly indicated that IVP calves had heavier birth weights
than IVD calves but that the average gestation length of IVP calves was not always longer than that of IVD calves. Furthermore, the birth rate of heavier calves and the incidence of stillbirth and
∗Corresponding author. Tel.:+81-229723101; fax:+81-229722326.
E-mail address: numabe@mtj.biglobe.ne.jp (T. Numabe).
perinatal mortality up to 48 h post partum in IVP calves (bull A: 11.3%, bull B: 7.8%) were greater (P <0.05) than those in IVD calves from both bulls (bull A: 4.1%, bull B: 3.7%). © 2000 Elsevier Science B.V. All rights reserved.
Keywords: Birth weight; Gestation length; In vitro culture; Cattle embryo
1. Introduction
It has been reported that in vitro matured and fertilized bovine oocytes can be developed to morula/blastocyst stage through co-culturing with bovine oviductal cells (Lu et al., 1988) and cumulus cells (Fukuda et al., 1990; Goto et al., 1988; Takada et al., 1991) or without co-culture cells (Bavister et al., 1992; McLanghlin et al., 1990; Pinyopummintr and Bavister, 1991; Rosenkrans and First, 1991). As well, the birth of normal calves from the transfer of in vitro produced (IVP) embryos has been reported (Fukuda et al., 1990; Goto et al., 1988; Lu et al., 1988; Takada et al., 1991). In Japan, Holstein dairy cattle are being used as recipients of IVP embryos for the purpose of increasing the number of Japanese Black cattle for meat production (Fukuda et al., 1990; Takada et al., 1991).
A high incidence of birth weights heavier than usual has been reported in calves conceived by transfer of IVP embryos (Behboodi et al., 1995; Kruip and den Daas, 1997; Young et al., 1998; Thompson et al., 1994, 1995; Walker et al., 1992, 1996; Wilson et al., 1995). In addition, calves produced by the transfer of embryos derived from in vitro cultures after nuclear transfer have been reported (Garry et al., 1996; Willadsen et al., 1991) to have heavier birth weights resulting from a pregnancy of long duration. In vitro culturing itself, the addition of serum in culture media (Walker et al., 1992), and the cells used in co-culturing during in vitro development can be suggested to be causal factors for these findings (Behboodi et al., 1995). However, a definitive explanation of the causes of heavier birth weight and longer gestation period has so far not been derived.
The aim of this study was to evaluate the difference in birth weight and gestation length between Japanese Black calves obtained from transfer of IVP and IVD embryos to Holstein recipients. An additional objective was to clarify the effects of sire on birth weight and gestation length and to examine the birth rate of heavier calves and the incidence of stillbirth in IVP and IVD calves.
2. Materials and methods
2.1. Sources of data
2.2. In vitro embryo production
Bovine ovaries were collected from Japanese Black cattle at a local slaughterhouse and transported to the laboratory in physiological saline maintained at 30–35◦C for 3 h. Cumulus-oocyte complexes (COCs) were collected by aspiration of follicles 2–8 mm in diameter using a 10 ml syringe attached to a 21 gauge needle. Only oocytes with intact, unexpanded, and compact cumulus cells were selected. All selected COCs were thoroughly washed twice in maturation medium; Hepes-buffered TCM 199 (Gibco BRL, Grand Island, NY, USA) supplemented with 5% heat-treated fetal bovine serum (FCS; Gibco BRL). After washing, groups of 40–50 COCs were placed in 500l of the maturation medium under min-eral oil (Squibb and Sons, Princeton, NJ, USA) in wells of a 4-well culture dish (NUNCLON; Nunc Co., Kampstrup, Denmark) and cultured for 20–22 h at 38.5◦C under 5% CO2in air.
For in vitro fertilization (IVF), one 0.5 ml straw of frozen semen (bull A or B) was used and the procedure of IVF was carried out as previously described (Takada et al., 1991). Bovine frozen-thawed spermatozoa were washed twice with BO medium (Brackett and Oliphant, 1975) supplemented with 10 mM caffeine (Caffeine Anhydrous, Wako Pure Chemical Industries, Osaka, Japan) by centrifugation at 450×gfor 10 min. The final sperm pellet was resuspended with the same medium to adjust to a concentration of 1.0–2.0×107 sperm/ml. An equal volume of BO medium supplemented with 3 mg/ml bovine serum albumin (BSA; A-4378, Sigma, St. Louis, MO, USA) and 10 IU/ml heparin (NOVO Heparin, NOVO Nordisk pharma Ltd, Bagsvaerd, Denmark) was added to the sperm suspension. The heparin-treated spermatozoa under mineral oil were incubated for 15 min at 38.5◦C under 5% CO2 in air. Groups of COCs (30–50) matured in vitro were introduced into 100l
microdrops of sperm suspension in a 35 mm culture dish (SUMILON; Sumitomo Bakelite Co., Tokyo, Japan).
After 6 h of post-insemination, the oocytes were washed and transferred to development medium; Hepes-buffered TCM 199 supplemented with 5% heat-treated calf serum (CS; Gibco BRL). Ten to twenty oocytes with cumulus cells were cultured in 50l microdrops of development medium covered with mineral oil (Squibb and Sons) at 38.5◦C under 5% CO2 in air. After 3 days of post-insemination, oocytes fertilized in vitro were assessed
morphologically for cleavage, and uncleaved oocytes were discarded from drops of the development medium. The cleaved embryos were cultured on a monolayer of cumulus cells up to Day 7 or 8.
2.3. In vivo embryo production
767 donors using a balloon catheter on Day 7 after the first detection of estrus and first insemination (Day 0).
2.4. Freezing and thawing of embryos
Either IVP or IVD embryos were placed in a 1.4 M glycerol and 0.2 M sucrose mixture in PBS supplemented with 20% CS (Massip et al., 1987; Ohisa et al., 1991), and equilibrated for 20–30 min at room temperature (to 25◦C). During equilibration, the embryos were loaded in-dividually with 0.1 ml of freezing medium into a 0.25 ml straw. In the straws, 0.1 ml of 0.3 M sucrose in PBS supplemented with 20% CS was loaded as a dilution solution and the end of the straw was sealed by heating. The straws were placed directly into the precooled chamber of a programmable freezer (ET-1, Fujihira Kogyo Co., Tokyo Japan) at−7◦C. After 2 min, the straws were seeded, maintained at−7◦C for 8 min, cooled to−23◦C at 0.5◦C/min and kept at−23◦C for 10 min before being plunged into liquid nitrogen (LN2). The straws were
maintained in LN2for 2–286 days. The straws were then thawed by maintaining them in air
for 5 s followed by immersion in a 35–38◦C water bath until all the ice disappeared. After 1 or 2 min, the cryoprotectant and 0.3 M sucrose were mixed within the straw which was placed vertically for 5 min. The frozen-thawed embryos were then transferred to the recipient cows.
2.5. Transfer of embryos
Embryos derived from Japanese Black cows were transferred to Holstein recipients on Day 7±1 of the estrous cycle. IVD embryos at the compact morula or blastocyct stage (Day 7) and of excellent or good quality were used for embryo transfer. On the other hand, IVP embryos at 7 or 8 days of post-insemination at the blastocyst stage and of excellent quality were selected. Each IVP or IVD embryo was washed several times in PBS supplemented with 20% CS and loaded into a 0.25 ml straw. Fresh embryos were transported to the dairy farms at 35◦C within 3 h. The frozen embryos were thawed at the dairy farms. Embryos were transferred non-surgically to the uterine horn ipsilateral to the ovary bearing the corpus luteum using a transverse hole-type transfer device (IVM; International Corp., Minneapolis, MN). Twenty-four technicians performed the transfer of embryos in this experiment. Pregnancy was diagnosed by fetal membrane palpation through rectal inspection at approximately Days 60–70 after estrus.
In this study, the rates of pregnancy and birth of IVP embryos (fresh and frozen) were 41.3 (426/1032) and 36.4% (376/1032). On the other hand, the rates of pregnancy and birth of IVD embryos (fresh and frozen) were 61.9 (618/998) and 58.7% (586/998). From these pregnancies, only data from single calves at birth were used. Out of 376 single calves, 48 (12.8%) were born by IVP twin transfers and out of 618 single calves, 49 (8.4%) were born by IVD twin transfers.
2.6. Statistical analysis
Table 1
Birth weight and gestation length of the calves by transfer of in vitro produced (IVP) and in vivo developed (IVD) bovine embryosa
aValues in the same column and sire group with different letters differ signficantly (P <0.01). bBull as semen sources.
3. Results
Birth weight of the IVP calves was significantly higher (P < 0.01) than that of IVD (Table 1). Gestation length of the IVP calves for bull A was significantly longer than that of IVD calves. However, for bull B, there was no difference in gestation length between IVP and IVD calves.
The birth rates of heavier IVP calves (>40 kg) were 9.8% for bull A and 7.8% for bull B within a gestation length of 290 days, and were 4.5 and 4.9%, irrespective of the gestation length. The birth rate of heavier IVP calves was significantly higher (P <0.05) than that of IVD calves regardless of the sire (Table 2).
Table 2
Percentage of calves exceeding 40 kg within 290 days of the gestation length and irrespective of gestation lengtha
Bullb Origin No. of calves Birth weight and gestation length 40 kg> ,<290 day (%)
aValues in the same column and sire group with different letters different significantly (P <0.05). bBull as semen sources.
Table 3
Stillbirth and pernatal motality incident rates of the calves by transfer of in vitro produced (IVP) and in vivo developed (IVD) bovine embryosa
Bullb Origin No. of calves No. of calves with stillbirth plusc(%)
A IVP 133 15 (11.3) a
IVD 121 5 ( 4.1) b
B IVP 243 19 (7.8) a
IVD 465 17 (3.7) b
aValues in the same column and sire group with different letters differ significantly (P <0.05). bBull as semen sources.
The incidence of stillbirth and perinatal mortality which occurred within 48 h post partum (stillbirth plus perinatal mortality) in IVP and IVD calves for both sires is shown in Table 3. The incidence rates of stillbirth plus perinatal mortality in IVP calves for bull A and B were 11.3 and 7.8%, respectively, and significantly greater than those in IVD (4.1 and 3.7%). It became evident from this result that calves with a heavy birth weight that were derived from IVP have a high accident rate at the time of birth.
4. Discussion
It was observed in this study that the birth weight of IVP calves was significantly heavier than that of IVD calves regardless of the bull. The gestation length of IVP calves was significantly longer than that of IVD calves for bull A. However, the gestation length of IVP calves was not always longer than the others and was affected by the bull. Therefore, the greater weights of calves are not due simply to longer gestation length. The results suggest that calves derived from IVP had a faster prenatal growth rate independent of gestation length. When IVP and IVD calves were compared for both bulls, the birth weights of IVP calves were heavier than those of IVD calves. This showed that regardless of the artificial process of transferring embryos, birth weight is affected by the origin of the embryo. Therefore, it is suggested that the technique of culturing in vitro embryos or maturation of oocytes is related to the heavier birth weight of IVP calves.
The birth rate of heavy IVP calves was 9.3 and 7.4% for bulls A and B, respectively, which was higher than that of IVD (1.7 and 1.3%) calves. Moreover, the incidence of stillborn IVP calves was higher than that of IVD calves (Table 2). From this study, differences in co-culturing cells and recipients as well as environmental factors and management of the recipients could be considered the causal factor of these findings. Therefore, the effect of in vitro culturing systems and the serum components of serum ought to be further investigated. Walker et al. (1996) have reported a link between an increase in birth weight and non-synchrony between the intrauterine membrane and fetus. Farin and Farin (1995) have reported that the non-synchrony between the recipient cow and embryo induces abnormal-ity in organs and the skeleton. In the present study, the transfer between different breeds (transfer of Japanese Black cattle embryos to Holstein recipients) and the large difference between the breeding environments of the Holstein and Japanese Black cows are thought to contribute to the calves heavy birth weights of calves. However, since in our study Holstein cows were the recipients in the IVD transfer, the breeding management and the animals were considered to be the same as those of the recipients of the IVP transfer. Furthermore, compared with the birth weight of AI calves derived from each bull used in this study (bull A: 27.3±0.1 kg, bull B: 28.2±0.2 kg), the birth weights of IVD calves was not heavy. Therefore, difference between breeds should not be taken as the sole cause of the calves heavy birth weights and long pregnancy durations.
vitro culturing in the sheep oviduct, calves produced by in vitro fertilization and in vitro co-culturing with oviductal epithelial cells have a high birth weight, but gestation length does not differ among those groups. In the present study, calves that were produced from the transfer of embryos derived from in vitro fertilization with co-culturing with cumulus had heavier birth weights than did IVD calves. The sire affected the gestation length of IVP calves. Our results showed that longer gestation length was not always associated with heavier birth weight.
The reason of the birth of heavier calves by transfer of IVP embryos is unclear. The expression of various gene transcripts differs between IVP and IVD embryos (Niemann and Wrenzycki, 2000). For example, gap junction protein Connexin 43 (C×43) transcripts were detected in bovine morulae and blastocysts derived from IVD, whereas IVP blastocysts and hacthed blastocysts expressed no mRNA for C×43 (Wrenzycki et al., 1996). Abnormal gene expressions of embryos might lead to an abnormal placental or fetal development. Further, the cell allocation to inner cell mass (ICM) and trophectoderm (TE) of IVP blastocysts is affected by culture system used (Van Soom et al., 1996). An abnormal ICM/TE ratio could lead to an imbalance in placental development (Leese et al., 1998). The elucidation on the cause of large calf syndrome will need more investigations.
The production of Japanese Black calves from Holstein dairy cattle by transfer of IVP embryos means that dairymen can produce both milk from Holstein herd and high-quality beef from Japanese Black cattle. Also, there is an increase in the income obtained from the sales of such beef calves.
The causal factor for large calves is considered to be the conditions of in vitro fertilization and in vitro culturing, and this constitutes a significant problem. Furthermore, in regard to the higher incidence of stillbirth among these cattle, there is a need for a detailed investigation of the individual immunological functions and anti-disease capability of IVP calves.
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