Birth of piglets after OPS vitrification and transfer of compacted morula stage embryos with intact zona pellucida

In swine, five to six days post-insemination, morulae and blastocysts are collected together after uterine flushing. The purpose of this study was to vitrify zona pellucida-intact morulae with Open Pulled Straw (OPS) technology and obtain piglets after transfer. Morulae (200) were vitrified after a two-step equilibration in ethylene glycol, dimethyl sulfoxide and sucrose in Hepes-buffered TCM199 + 20% NBCS medium (TCM). 2-6 morulae were loaded into OPS and plunged into liquid nitrogen. At embryo warming, a three-step dilution with decreasing concentrations of sucrose was applied. In each of 10 recipients, 20 morulae were transferred surgically. Day 25, gestation rate and the farrowing rate were 80% and 70%, respectively. The pregnant recipients farrowed from 1 to 8 piglets and the survival of total transferred embryos was 13%. Although survival rates are still compromised, OPS technology is therefore appropriate to cryopreserve porcine morulae with intact zona pellucida.     

Changes to porcine blastocyst vitrification methods and improved litter size after transfer

The objective was to improve the protocol that was used to obtain the first reported piglets from transferred vitrified and warmed zona-intact blastocysts. Blastocysts were collected from superovulated sows and gilts, centrifuged to polarize lipid, vitrified, warmed and cultured for 24h or transferred immediately. Removing the zona pellucida after warming increased the number of cells in the surviving blastocysts (zona-free 60.8+/-4.3, zona-intact 39.1+/-2.8; P<0.05). Thinning the zona pellucida produced similar results to zona removal. Changing the basal medium of the vitrification and warming solutions from modified PBS to phosphate buffered NCSU-23 increased the number of cells (44.7+/-2.2 versus 56.0+/-3.9, respectively; P<0.05). Reducing the plunge temperature of the liquid nitrogen from -196 degrees C to less than -204 degrees C improved the embryo survival rate (61.9% versus 82.9%, respectively; P<0.05). These modifications were incorporated into the vitrification protocol that was used to vitrify and warm 105 blastocysts (that were subsequently transferred into four recipients). Three recipients became pregnant, farrowing three litters (average litter size, 5.3; 18.8% embryo survival in farrowing sows). Changing the warming protocol to using sucrose rather than ethylene glycol resulted in a trend towards improved embryo survival (73.5% versus 91.2%) but this was not statistically significant. Incorporating this modification, 203 blastocysts were vitrified, warmed and transferred into seven recipients. Five became pregnant and 36 fetuses were recovered (average litter size 7.2; 24.8% embryo survival in pregnant sows) at Day 40 of pregnancy. In conclusion, changes made to the vitrification protocol improved pregnancy rate and in vivo embryo survival compared to an earlier study using the original protocol.     

Effect of birth litter size, birth parity number, growth rate, backfat thickness and age at first mating of gilts on their reproductive performance as sows

The present study was performed to evaluate retrospectively the influence of birth litter size, birth parity number, performance test parameters (growth rate from birth to 100kg body weight and backfat thickness at 100kg body weight) and age at first mating (AFM) of gilts on their reproductive performance as sows. Traits analysed included remating rate in gilts (RRG), litter size, weaning-to-first-service interval (WSI), remating rate in sows and farrowing rate (FR). Data were collected from 11 Swedish Landrace (L) and 8 Swedish Yorkshire (Y) nucleus herds and included 20712 farrowing records from sow parities 1-5. Sows that farrowed for the first time during 1993-1997, having complete records of performance test and AFM, were followed up to investigate their subsequent reproductive performance until their last farrowing in 1999. Analysis of variance and multiple regression were applied to continuous data. Logistic regression was applied to categorical data. The analyses were based on the same animals and the records were split into six groups of females, i.e. gilts, primiparous sows, and sows in parities 2-5, respectively. Each additional piglet in the litter in which the gilt was born was associated with an increase of her own litter size of between 0.07 and 0.1 piglets per litter (P<0.001). Gilts born from sow parity 1 had a longer WSI as primiparous sows compared with gilts born from sow parity 4 (0.3 days; P<0.05) or parity 5 (0.4 days; P<0.01). Gilts with a higher growth rate of up to 100kg body weight had a larger litter size (all parities 1-5; P<0.05), shorter WSI (all parities 1-5; P<0.05) and higher FR (parities 2 and 5; P<0.05) than gilts with a lower growth rate. Gilts with a high backfat thickness at 100kg body weight had a shorter WSI as primiparous sows (P<0.001) compared with low backfat gilts, and 0.1 piglets per litter more as second parity sows (P<0.01). A 10 day increase in AFM resulted in an increase in litter size of about 0.1 piglet for primiparous sows (P<0.001) and a decrease (P<0.05) for sow parities 4 and 5.     

A procedure for successful nonsurgical embryo transfer in swine

The current study was undertaken to develop a successful procedure for the nonsurgical transfer of pig embryos. A total of 663 embryos were surgically collected on Day 4 or 5 from 55 donors, of which 542 embryos of acceptable quality were nonsurgically transferred to 46 recipients. Nonsurgical recipient gilts were sedated 15 min prior to transfer with 20 mg im acepromazine maleate. A disposable insemination spirette with an attached 3-way stopcock was manipulated into the cervix of each gilt. Embryos were expelled from a tomcat catheter into the spirette, and 10 to 12 ml of Whitten's medium were used to flush embryos through the spirette into the reproductive tract. Sixteen (34.8%) recipient gilts did not return to estrus before Day 36, and 10 (21.7%) gilts farrowed with an average litter size of 4.3 +/- 0.7. Embryos were collected from an additional 20 donors and were surgically transferred to an additional 19 recipients. Surgical transfers conducted at the same time as the nonsurgical transfers resulted in 12 (63.2%) gilts farrowing and 7.1 +/- 0.6 pigs were born per litter. In conclusion, a procedure has been developed for nonsurgical transfer of swine embryos which simplifies the process of embryo transfer and which may increase the potential for utilization of embryo transfer technologies by swine producers.     

猪胚胎开放式拉长细管玻璃化冷冻保存研究

从20头供体母猪获得的291枚可用胚胎(囊胚/桑葚胚),采用二步法开放式拉长细管(OPS,openpulledstraw)玻璃化冷冻技术进行保存,即胚胎首先在冷冻液I(TCM199 20?S 10%EG 10%DMSO)中平衡3min,然后立即转入冷冻液II(TCM199 20?S 20%EG 20%DMSO 0.4mol/LSUC)中并在1min内装管,直接投入液氮保存;3个月后解冻移植给8头受体母猪,其中1头怀孕产仔(8头活仔),在我国首次获得猪胚胎超低温(-196℃)冷冻后代。     

An association analysis of sow parity,live-weight and back-fat depth as indicators of sow productivity

Understanding how critical sow live-weight and back-fat depth during gestation are in ensuring optimum sow productivity is important. The objective of this study was to quantify the association between sow parity, live-weight and back-fat depth during gestation with subsequent sow reproductive performance. Records of 1058 sows and 13 827 piglets from 10 trials on two research farms between the years 2005 and 2015 were analysed. Sows ranged from parity 1 to 6 with the number of sows per parity distributed as follows: 232, 277, 180, 131, 132 and 106, respectively. Variables that were analysed included total born (TB), born alive (BA), piglet birth weight (BtWT), pre-weaning mortality (PWM), piglet wean weight (WnWT), number of piglets weaned (Wn), wean to service interval (WSI), piglets born alive in subsequent farrowing and sow lactation feed intake. Calculated variables included the within-litter CV in birth weight (LtV), pre-weaning growth rate per litter (PWG), total litter gain (TLG), lactation efficiency and litter size reared after cross-fostering. Data were analysed using linear mixed models accounting for covariance among records. Third and fourth parity sows had more (P<0.05) TB, BA and heavier BtWT compared with gilts and parity 6 sow contemporaries. Parities 2 and 3 sows weaned more (P<0.05) piglets than older sows. These piglets had heavier (P<0.05) birth weights than those from gilt litters. LtV and PWM were greater (P<0.01) in litters born to parity 5 sows than those born to younger sows. Sow live-weight and back-fat depth at service, days 25 and 50 of gestation were not associated with TB, BA, BtWT, LtV, PWG, WnWT or lactation efficiency (P>0.05). Heavier sow live-weight throughout gestation was associated with an increase in PWM (P<0.01) and reduced Wn and lactation feed intake (P<0.05). Deeper back-fat in late gestation was associated with fewer (P<0.05) BA but heavier (P<0.05) BtWT, whereas deeper back-fat depth throughout gestation was associated with reduced (P<0.01) lactation feed intake. Sow back-fat depth was not associated with LtV, PWG, TLG, WSI or piglets born alive in subsequent farrowing (P>0.05). In conclusion, this study showed that sow parity, live-weight and back-fat depth can be used as indicators of reproductive performance. In addition, this study also provides validation for future development of a benchmarking tool to monitor and improve the productivity of modern sow herd.     

The influence of time of insemination relative to time of ovulation on farrowing frequency and litter size in sows, as investigated by ultrasonography

The objective of this experiment was to identify the optimal time of insemination relative to the time of ovulation, based on ultrasonographic detection of embryonic survival at 10 days after ovulation, number of sows farrowing, and litter size. Furthermore, the possible value of the interval from weaning to onset of estrus for prediction of the time of ovulation was examined. Crossbred sows (n = 143) that had farrowed 2 to 9 litters were weaned (Day 0) and observed for estrus every 8 h from Day 3 until end of estrus. Ultrasonography was performed every 6 h, from 12 h after onset of estrus until ovulation had been observed. The sows were inseminated once at various time intervals from ovulation. At Day 16, 25 of the sows were slaughtered and their uteri were flushed for embryos. In the remaining sows, the number of viable and dead piglets and mummified fetuses per sow was recorded at farrowing, with the sum of the 3 constituting the total number of piglets born per sow. The highest number of embryos recovered per sow was found after insemination during the interval from 24 h before to 4 h after ovulation. The lowest frequency of non-pregnant sows and the highest total number of piglets born per sow were found after insemination from 28 h before to 4 h after ovulation. Consequently, the optimal time for insemination was found to be in the interval 28 h before to 4 h after ovulation. The interval from weaning to onset of estrus and from onset of estrus to ovulation were negatively correlated, allowing a rough prediction of the time of ovulation from the interval from weaning to onset of estrus.     

Production of piglets preselected for sex following in vitro fertilization with X and Y chromosome-bearing spermatozoa sorted by flow cytometry

In vivo-matured porcine oocytes were fertilized in vitro with X and Y chromosome-bearing spermatozoa, and sorted for sex on the basis of DNA content by flow cytometry. Developmental competence of the sexed embryos was determined through established pregnancies after embryo transfer. Spermatozoa were stained with Hoechst 33342 and sorted using a flow cytometry cell sorter. Purity of sorting was 83% for Y spermatozoa and 92% for X spermatozoa. A total of 387 mature cumulus-oocyte-complexes (COC) was collected from 18 superovulated prepuberal gilts shortly before ovulation. In vitro fertilization with sorted spermatozoa was performed in 4 replicates. After 18 h of sperm- oocyte co-culture at 39 degrees C, the zygotes were placed into culture medium (NCSU-23) for another 24 h. The average cleavage rate was 56.2%. Ninety-two embryos produced from X-sorted sperm cells were transferred surgically into the uterus of 2 recipients. Two gilts farrowed and delivered 6 and 4 healthy female piglets, respectively. Additionally, 2 gilts were inseminated intratubally via surgical laparotomy with either X or Y sorted spermatozoa (2 x 10(5)) per oviduct. The 2 sows farrowed producing 15 piglets. Thirteen of the 15 piglets were of the predicted gender (85%).     

猪胚胎玻璃化冷冻保存技术的优化

在以往工作的基础上本研究对现有猪胚胎玻璃化冷冻保存技术进行了优化。以巴马小型猪为供体,采用超数排卵技术,采集5~6日龄的胚胎(囊胚/桑椹胚),比较冷冻方法、胚胎承载工具胚胎、透明带处理和冷冻胚胎移植受体对猪胚胎冷冻效果的影响。结果表明,冷冻方法I[胚胎首先在冷冻液1(TCM199+20%FBS+10%EG+10%DMSO)中平衡3min,然后立即转入冷冻液2(TCM199+20%FBS+20%EG+20%DMSO+0.4mol/LSUC)中并在1min内装管(每管含2~6枚胚胎),直接投入液氮保存]与冷冻方法II[透明带完整的胚胎在NCSU23(含7.5μg/mLCB)培养液中平衡25min,13000×g离心12~13min,然后在含2mol/LEG的NCSU23中平衡5min,再在含8mol/LEG+7%PVP的NCSU23中快速漂洗,装进OPS/GMP管,放入液氮保存]冷冻效果没有显著差异;GMP法能显著提高冷冻胚胎存活率(83.8%vs77.6%,P<0.05)和囊胚细胞数(47.5vs53.1,P<0.05);以0.5%链蛋白酶(Pronase)处理透明带10s,虽然对猪胚胎存活率没有显著影响,但能...     

Successful nonsurgical deep uterine embryo transfer in pigs

At present, it is possible to transfer pig embryos directly into the uterine body of sows by nonsurgical procedures. The aim of this study was to develop a procedure for nonsurgical embryo transfer (ET) into the upper part of one uterine horn in gilts and sows. In experiment 1, 29 gilts and 43 sows were used. Intrauterine insertions took place for each female at days 4-6 of the estrous cycle (D0 = onset of estrus). An artificial insemination (AI) spirette was inserted into the cervix to assist with the guidance of a modified flexible catheter originally developed for deep intrauterine insemination in pigs. The flexible catheter length inserted anterior to the inserted AI spirette was 43.0 +/- 1.7 cm. The time required to complete the procedure was affected by the type of female (P < 0.001) and by the difficulties encountered for inserting the catheter (P < 0.001). However, when no or minor difficulties were encountered during the insertion of the catheter (in approximately 70 and 80% of gilts and sows, respectively), the time required to complete the procedure did not differ between gilts (2.5 +/- 0.1 min) and sows (2.3 +/- 0.1 min). In experiment 2, 24 to 31 fresh morulae and/or blastocysts were transferred to each of 24 recipients. Seventeen animals (70.8%) farrowed an average of 6.9 +/- 0.7 piglets, of which 0.6 +/- 0.3 piglets were born dead. In conclusion, the procedure described in this study offers new possibilities to transfer embryos nonsurgically to the uterine horn of pigs.     

Herd-level factors associated with piglet weight at weaning,kilograms of piglets weaned per sow per year and sow feed conversion

To understand the production factors that affect conclusive parameters of sow herd performance can improve the use of the resources and profitability of farm. The objective of this study was to identify associations and quantify the effects of a set of factors related to piglet weight at weaning (PWW), kilograms of piglets weaned per sow per year (kgPWSY) and sow feed conversion (SFC). Data from 150 farms were collected, for a total study population of 135 168 sows, including gilt replacement, breeding (mating), gestation and farrowing/lactation phases. A questionnaire focusing on reproductive performance, management, facilities, feeding, health and biosafety was administered. Multiple linear regression models were used to assess associations among factors with each of the three dependent variables. Increased duration of lactation was positively associated with PWW, kgPWSY and SFC. The increase in the number of live born pigs per litter was positively associated with kgPWSY and with SFC. Farms with higher PWW had farrowing room humidifiers, did not surgically castrate male piglets and used quaternary ammonia compounds for farrowing room disinfection. Farms with higher kgPWSY used lined ceilings in farrowing rooms and winter feeds with higher CP percentages in gestation; they also had more farrowings per sow per year. Sow feed conversion was worse in farms with partly slatted floors during gestation, in farms feeding lactating sows six times a day or ad libitum and farms with a higher sow-handler ratio. This study indicates that farms can increase PWW and kgPWSY and improve the SFC by changing one or more management, biosafety and feeding practices or facilities as well as by focusing on improving several performance parameters, particularly increasing the duration of lactation and the number of live born pigs per litter.     

A behavioural scale to measure ease of farrowing in sows

Farrowing is one of the most critical phases in pig production, as it has an impact on neonatal pig survival. Assessing the ease of farrowing can improve the management of sows and thus increase litter survival. The aims of this study were: 1) to develop an ease of farrowing score (EFS) in sows based on the behaviour of the sows and their piglets, and 2) to determine the relationship between the EFS and productive, physiological, and subjective parameters. Eighty hybrid (Large White × Landrace) sows from first to seventh parity housed in individual crates were used. An EFS was constructed using the total duration of farrowing, the birth interval, the total time standing or sitting, the number of position changes during the day before and the day of farrowing, the sow posture at birth, the viability and the position of the piglets at birth (head or back born). Moreover, rectal temperature at 90 min after farrowing, a four categorical subjective visual assessment (VA) of farrowing and litter size (piglets born alive, stillborn and mummified foetus) were recorded. A common factor analysis model yielded five factors with an eigenvalue higher than 0.95 that accounted for 75.05% of the total variation between individuals. The three main factors were “farrowing duration”, “sow posture”, and “sow activity” of sows and explained 23.44%, 15.67%, and 14.23% of the variance, respectively. Primiparous sows had higher values for factor 3 (sow activity) than multiparous sows (P = 0.02). Sows without stillborn or mummification foetus showed higher values of EFS than sows with at least one stillborn or mummification foetus (P = 0.06 and 0.01, respectively). Sows that received a visual assessment of 3 and 4 showed higher values of EFS than sows that received a VA of 1 and 2 (P = 0.0017). The EFS appears to be a good behavioural scale to measure ease of farrowing in sows kept in individual farrowing crates. Duration of farrowing, sow position, and presence of stillborn piglets and mummified foetuses appear to be important ease of farrowing indicators.     

Piglets born after non-surgical deep intrauterine transfer of vitrified blastocysts in gilts

The aims of this study were: (1) to evaluate the effect of the number of previous estrus of recipient gilts on effectiveness of intrauterine insertion of a flexible catheter designed for non-surgical deep intrauterine catheterization during diestrus in pigs; and (2) to determine the farrowing rate and the litter size after non-surgical deep intrauterine embryo transfer (ET) of porcine blastocysts vitrified by the open pulled straw (OPS) method. In experiment 1, 27 large white hyperprolific gilts (LWh) with 2-6 previous estrus were used. Intrauterine insertions of the flexible catheter were carried out at day 5.5-6 of the estrous cycle (D0=onset of estrus). During insertions, no or only moderate reactions were observed in 88.9% of gilts and was not related (P >0.05) to the number of estrus prior to the insertion periods. The number of the estrus had a significant effect (P <0.05) on the difficulties found during the procedure. In the 100% of gilts with two estrus (N=6) it was not possible to insert the flexible catheter through the cervix. In gilts with three or more estrus, it was possible to pass the cervix and to progress along a uterine horn in 80.9% of the cases. In 86.7% of the gilts, the tip of the flexible catheter achieved the second or third quarter of the uterine horn. In experiment 2, following non-surgical deep intrauterine transfer of 20 vitrified/warmed blastocysts, 9 Meishan recipients (42.9%) farrowed an average of 5.4 +/- 0.8 piglets (range 3-9) of which 0.6 +/- 0.3 piglets (range 0-2) were born dead. In conclusion, this study shows that it is possible to obtain birth of piglets following non-surgical deep intrauterine embryo transfer (ET) of vitrified/warmed blastocysts. Non-surgical deep intrauterine ET and OPS vitrification methods are promising procedures to be used together for the introduction of new genetic material in a farm.     

Effect of insemination of estrus-induced prepuberal gilts on ensuing reproductive performance and body weight

Prepuberal gilts reared and managed to 85-90 kg live weight in a common system were allocated at random to one of three first-mating treatments in an experiment conducted over a period of more than 5 years. In two of the treatments, gilts received a single i.m. injection of 400 IU equine chorionic gonadotropin (eCG) and 200 IU human chorionic gonadotropin (hCG) (PG600; Intervet) and were either inseminated 4 and 5 days later on a fixed-time basis regardless of oestrus (treatment A), or at the second oestrus following treatment (treatment B). The third group of gilts remained untreated and was inseminated on the first spontaneous oestrus (treatment C). Thereafter, all gilts were managed in the same way and those observed in oestrus were re-inseminated. Significantly more gilts returned to oestrus after the first service in treatment A (35%) than in treatment B and C (12 and 17%, respectively; P<0.01). Gilts farrowed to the first or repeat inseminations at a significantly younger age (P<0.01) in treatment A (304 days) than treatment B (324 days) and C (320 days). The age difference at farrowing remained in surviving gilts at the end of their third parity. The first farrowing performance of the gilts was significantly affected by treatment in terms of litter size at birth (A 7.0, B 8.4 and C 8.3 live piglets per gilt; P<0.01), litter size at weaning (A 6.2, B 7.2 and C 7.2 live piglets per gilt, P<0.05), and piglet birth weight (A 1.4, B 1.3 and C 1.3 kg; P<0.05) but piglet survival rate and weaning weight were not affected by treatment. The live weights of the gilts were significantly different between the treatments at first insemination (A 95.7, B 106.5 and C 109.2 kg; P<0.01) but not when the first litter was weaned (A 133.6, B 135.1 and C 136.6; P>0.05). After the first farrowing there were no differences between the treatments in terms of the survival rate, productive or reproductive performance of the gilts/sows and their offspring. Without conducting a detailed cost-benefit-calculation it was deduced that, from an economical point of view, differences between treatment A and treatments B and C are negligible because the savings associated with farrowing at a younger age on this treatment just about compensated for any additional costs associated with the treatment and the lower number of piglets born at the first farrowing.     

Piglets born from vitrified cloned blastocysts produced with a simplified method of delipation and nuclear transfer

Successful cryopreservation of porcine embryos offers a promising perspective in the fields of agriculture, animal science, and human medical research. The objective of the present work was to establish a system facilitating the cryopreservation of porcine embryos produced by somatic cell nuclear transfer (SCNT). Several key techniques including micromanipulator-based enucleation, noninvasive delipation, zona-free fusion, and activation were combined with high efficiency. After a partial zona digestion and high-speed centrifugation, 89.8+/-2.1% (mean+/-SEM) of enucleated oocytes were successfully delipated. Delipated cytoplasts were incubated for an additional 0.5 or 2 h before fusion with somatic cells. After activation and 6 days of in vitro culture, no significant difference in the rate of blastocysts per reconstructed embryo was observed between the two groups (33.1+/-1.8% and 26.0+/-4.3% for 0.5 and 2 h recovery time, respectively). Cryopreservation of the blastocysts was performed with a Cryotop device and factory-prepared vitrification and warming solutions. One hundred fifty-five vitrified SCNT embryos were transferred surgically into two recipient sows to test their developmental capacity in vivo. One recipient became pregnant and delivered six piglets. In conclusion, our simplified delipation and SCNT procedure resulted in viable piglets after vitrification and embryo transfer at the blastocyst stage.     

Confinement of lactating sows in crates for 4 days after farrowing reduces piglet mortality

To reduce mortality among suckling piglets, lactating sows are traditionally housed in farrowing crates. Alternatively, lactating sows can be housed in farrowing pens where the sow is loose to ensure more behavioural freedom and consequently a better welfare for the sow, although under commercial conditions, farrowing pens have been associated with increased piglet mortality. Most suckling piglets that die do so within the first week of life, so potentially lactating sows do not have to be restrained during the entire lactation period. Therefore, the aim of the current study was to investigate whether confinement of the sow for a limited number of days after farrowing would affect piglet mortality. A total of 210 sows (Danish Landrace × Danish Yorkshire) were farrowed in specially designed swing-aside combination farrowing pens measuring 2.6 m × 1.8 m (combi-pen), where the sows could be kept loose or in a crate. The sows were either: (a) loose during the entire experimental period, (b) crated from days 0 to 4 postpartum, (c) crated from days 0 to 7 postpartum or (d) crated from introduction to the farrowing pen to day 7 postpartum. The sows and their subsequent litters were studied from introduction to the combi-pen ∼1 week before expected farrowing and until 10 days postpartum. Confinement period of the sow failed to affect the number of stillborn piglets; however, sows that were crated after farrowing had fewer live-born mortality deaths (P < 0.001) compared with the sows that were loose during the experimental period. The increased piglet mortality among the loose sows was because of higher mortality in the first 4 days after farrowing. In conclusion, the current study demonstrated that crating the sow for 4 days postpartum was sufficient to reduce piglet mortality.     

Embryo survival and conceptus growth after reciprocal embryo transfer between Chinese Meishan and Landrace x Large White gilts

Embryos were transferred between Meishan and Landrace x Large White (control) gilts on Day 4 or 5 to establish approximately equal numbers of all four possible combinations of donor breed and recipient breed. The breed of the donor gilt significantly (P less than 0.01) affected embryo survival with 44.5% of transferred Meishan embryos and 69.6% of transferred control embryos surviving to Day 30 +/- 1. There was no influence of the breed of the recipient gilt on the proportion of embryos which survived. These differences in embryo survival between the two breeds could not be explained by differences in (1) the number of embryos transferred, (2) the stage of development of the embryos transferred, (3) the interval between ovulation and transfer or (4) the degree of asynchrony between donor and recipient gilt. On Day 30 +/- 1 embryos from control donors developed into longer fetuses (P less than 0.01) with larger allantoic sacs (P less than 0.05) than did embryos from Meishan donors. Fetuses in control recipients were longer (P less than 0.01), heavier (P less than 0.001) and had larger allantoic sacs (P less than 0.05) than fetuses occupying Meishan uteri. The interaction between breed of donor gilt and breed of recipient gilt did not significantly affect conceptus growth. These results suggest that Meishan pig embryos may be less tolerant to routine embryo transfer procedures than those of control gilts, that the genotype of the dam does not affect the proportion of embryos surviving to Day 30 +/- 1, and that both fetal and maternal factors affect conceptus growth.     

Successful production of piglets derived from vitrified morulae and early blastocysts using a microdroplet method

This study was conducted to determine the efficiency of vitrification using the microdroplet (MD) method for early stage porcine embryos. Embryos at compacted morulae to early blastocyst stage were vitrified in a vitrification solution containing 40% (v/v) ethylene glycol, 0.6M sucrose and 2% (w/v) polyethylene glycol in M2 (ESP) without any pretreatment. The equilibration and dilution were carried out in third and fourth steps, respectively, at 38 degrees C. The survivability of the cryopreserved embryos was assessed for both in vitro culture (Experiment 1) and by embryo transfer (Experiment 2). In Experiment 1, the embryos were vitrified within a microdroplet or 0.25 ml straw (ST) and fresh embryos were used as a control group. The survival rates after 24h culture in the MD, ST and control groups were 21/23, 14/20 and 20/20, respectively. The hatching rates of the embryos after 48 h incubation were 14/23, 4/20 and 16/20, respectively. In Experiment 2, 171 vitrified embryos were transferred to 5 recipient gilts, and 17 healthy piglets were produced from 2 recipients (3 recipients aborted) in Group 1. In Group 2, 81 vitrified embryos and 16 fresh embryos in total were transferred to 4 recipient gilts, and 10 healthy piglets from the vitrified embryos were produced from 3 recipients. These results indicated that porcine embryos of compacted morulae to early blastocyst stage can survive cryopreservation using the microdroplet method without any special intracellular manipulation or treatment.     

Developmental competence of pig oocytes matured and fertilized in vitro

Pig follicles 3 to 6 mm in diameter were everted and matured for 44 h. The oocytes were then collected and exposed to capacitated boar sperm purified by centrifugation in a two step (65 and 70%) Percoll gradient. Of 110 ova fixed 14 h after in vitro fertilization, 78% were penetrated and 47% were monospermic. Next, 681 oocytes were cultured in vitro for 44 h after in vitro fertilization and the 266 embryos which had reached the two- to four-cell stage were transferred into the oviducts of 12 synchronized recipient gilts. Four days later, 211 embryos (79%) were recovered by uterine flushing. 40.7% of these were at the blastocyst stage, and 20% were at the morula stage. In a final experiment, four out of eight gilts which had received 40 to 50 two- to four-cell embryos, were diagnosed pregnant 30 and 37 d after in vitro fertilization. One sow farrowed nine live piglets and one stillborn, two pregnancies were in progress, while one sow returned to estrus 47 d after in vitro fertilization. These results demonstrate that pig oocytes matured and fertilized in vitro can develop to the blastocyst stage and establish a normal pregnancy resulting in the birth of live piglets.     

Production of nuclear transfer-derived piglets using porcine fetal fibroblasts transfected with the enhanced green fluorescent protein

A system for somatic cell nuclear transfer (SCNT) was developed and led to the successful production of GFP-transfected piglets. In experiment 1, two groups of SCNT couplets reconstructed with porcine fetal fibroblasts (PFF) and enucleated sow (S) or gilt oocytes (G): 1). received a simultaneous electrical fusion/activation (S-EFA or G-EFA groups), or 2). were electrically fused followed by activation with ionomycin (S-EFIA or G-EFIA groups), or 3). were subjected to electrical fusion and subsequent activation by ionomycin, followed by 6-dimethylaminopurine treatment (S-EFIAD or G-EFIAD groups). The frequency of blastocyst formation was significantly higher in S-EFA (26%) compared with that observed in the other experimental groups (P < 0.05), but not with S-EFIA (23%). Sow oocytes yielded significantly higher cleavage frequencies (68%-69%) and total cell numbers of blastocysts when compared with gilt oocytes, regardless of fusion/activation methods (P < 0.05). However, the ratio of inner cell mass (ICM)/total cells in G-EFA and S-EFA was significantly lower than in the other groups (P < 0.05). In experiment 2, SCNT couplets reconstructed with PFF cultured in the presence or absence of serum and enucleated sow oocytes were subjected to EFA. There were no effects of serum starvation on cell-cycle synchronization, developmental competence, total cell numbers, and ratio of ICM/total cells. In experiment 3, SCNT couplets reconstructed with PFF transfected with an enhanced green fluorescence protein (EGFP) gene using FuGENE-6 and enucleated sow oocytes were subjected to EFA and cultured for 7 days. Expression frequencies of GFP gene during development were 100%, 78%, 72%, 71%, and 70% in fused, two-cell, four to eight cells, morulae, and blastocysts, respectively. In experiment 4, SCNT embryos derived from different recipient cytoplasts (sows or gilts) and donor karyoplasts (PFF or GFP-transfected) were subjected to EFA and transferred to the oviducts of surrogates. The pregnancy rates in SCNT embryos derived from sow oocytes (66%-69%) were higher than those with gilt oocytes (23%-27%) regardless of donor cell types. One live offspring from GFP-SCNT embryos and two from PFF-SCNT embryos were delivered. Microsatellite analysis confirmed that the clones were genetically identical to the donor cells and polymerase chain reaction (PCR) from genomic DNA of cloned piglets and subsequent southern blot analysis confirmed the integration of EGFP gene into chromosomes.