Effects of different reproduction techniques: AI, moet or IVP, on health and welfare of bovine offspring

!I ELSEVIER

EFFECTS OF DIFFERENT REPRODUCTION TECHNIQUES: AI, MOET OR IVP, ON HEALTH AND WELFARE OF BOVINE OFFSPRING 1

A.M. van Wagtendonk-de Leeuw'a, E. MUllaart , A.P.W. de RoosJ. lS. Merton', l lH.G. den Daas , B. Kemp2 and 1. de Ruigh l tHolland Genetics, R&D, PO Box, 5073, 6802 EB Amhem, The Netherlands 2Wageningen Institute of Animal Science, Wageningen Agricultural University, The Netherlands ABSTRACT Since the introduction of in vitro production (IVP) of bovine and sheep pre-implantation embryos, increased birth weights and other deviations of IVP calves and lambs compared with AI or MOET offspring have been reported. Study I of the present paper, a comparison between AI, MOET and IVP (co-culture/serum) calves with respect to calf and calving characteristics in largescale field conditions, confirms these reports. In addition, it is shown that MOET calves tend towards higher birth weights and have significantly longer gestations and more difficult calvings than AI calves. It is presently unknown ifthe effect ofIVP is also observed later in life. In this paper, data on reproduction characteristics ofbovine IVP co-culture/serum offspring are presented. Semen production - and non return data of one year old IVP bulls and superovulation-, AI- and OPUJIVP results of one year old IVP heifers are compared with those of one year old AI and MOET animals producing semen or embryos in the same time period. So far, there are no indications that the use oflVP is reflected in deviate reproduction characteristics of bovine IVP offspring. It has been suggested that use of co-culture cells and serum during in vitro culture of bovine (and sheep) embryos may partially explain the increased birth weights and other deviations ofbovine and sheep IVP offspring. Deletion of these factors in semi-defined culture media, e.g. Synthetic Oviductal Fluid (SOF), could result in more normal offspring. Study 2 investigates this hypothesis in both field conditions (Study 2a, comparing AI, IVP co-culture/serum and IVP SOF calves) and in semi-standardized conditions (Study 2b, comparing MOET, IVP co-culture/serum and IVP SOF calves at one herd). In Study 2a, although IVP SOF calves showed (non-significant) shorter gestations, easier calvings and lower percentages of perinatal mortality and congenital malformations than IVP co-culture calves, birth weights were not decreased. In Study 2b however, the difference between IVP co-culture and IVP SOF calves in birth weight and ease of calving was significant (P<0.05), IVP SOF calves resembling MOET calves more. IVP calves differed significantly from MOET calves with respect to several physiological parameters, such as blood oxygen saturation level, heart beat frequency and some measures of the heart. In addition, in Study 2b, recipients receiving an IVP SOF embryo showed a more regular return to estrus than those receiving an IVP co-culture embryo. From Study 2 it can be concluded that using a semi-defined medium for in vitro culture (SOF) may improve characteristics of IVP calves born.

e 1999 by Elsevier Science Inc.

Key words: bovine IVP, co-culture/serum, SOF, birth weight, calving data, reproduction later in life Acknowledgments We thank Helga Flapper and Anne de Glee for ultrasonography of heart and kidneys; Rienk and Harry Waaksma for measurements on the calves; Dr. T. Wensing and Dr. J Vermeiden and his group for fruitful discussions; Dr. IF. Hasler for critical review of the manuscript; and all Holland Genetics staff participating in the IVP field trials for their excellent teamwork. aReprint requests to A.M. vanWagtendonk-de Leeuw, e-mail [email protected] Theriogenology 53:575-597, 2000

C 1999 by Elsevier Science Inc.

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Theriogenology

576 INTRODUCTION

During the last decades tremendous advances have been made in both human and animal assisted reproduction. In cattle, it started with the first calves born after artificial insemination at the start of this century. The next step was superovulation and embryo transfer in the early 1970s, followed by in vitro maturation, fertilization and culture in 1987 (5). The first child born after in vitro fertilization (IVF) in 1978 was Louise Brown (2) and, most recently in sheep, the first somatic cell cloned animal "Dolly" was born in 1997 (39). By now, most of these assisted reproduction methods have proven their advantages. In the human, the main advantage is overcoming reproductive problems and in animals, the main advantage is the effective use and dissemination of selected male and female genes in breeding programs. A prerequisite for continued successful application of an assisted reproduction method is the delivery of healthy "normal" individuals. The "Large Offspring Syndrome" Since the introduction of in vitro production (IVP) of embryos in animal reproduction, several studies have reported the so-called "Large Offspring Syndrome". In vitro produced ruminant embryos develop faster and are both morphologically (more lipid-like inclusions and less compaction) and functionally different from in vivo produced embryos (30). Transferred IVP embryos show reduced pregnancy rates and irregular returns to estrus compared to in vivo produced embryos. Last but not least, IVP embryos result in both calves and lambs that have an increased birth weight (4,11,36,38), a longer gestation period (14,26), an increased incidence of abortions (11), a higher perinatal mortality (1,14,36), more congenital abnormalities (25), more hydro-allantois (11,36), relatively more bull calves and more problems during calving compared to calves born after artificial insemination (AI) or after multiple ovulation and embryo transfer (MOET). Upon cloning (nuclear transfer), these problems seem to be even more pronounced: calves often show breath problems and symptoms of acidosis, are lethargic, slow and do not suckle spontaneously immediately after birth (8,40). For some recent reviews on the "Large Offspring Syndrome", see (11,14,15,41). In contrast to the bovine and sheep, human IVF children are generally lighter than their naturally conceived counterparts at birth and women pregnant from IVF embryos have a shorter gestation length (19). Analogous to cattle and sheep, abortion rates and perinatal mortality are increased. The IVF system in the human is different from the IVP system in cattle and sheep. Firstly, in the human, oocytes usually are collected from less fertile, or women with advanced maternal age vs in the bovine fertile, young animals. Secondly, maturation in the human is performed in vivo after superstimulation, whereas in the bovine immature oocytes, collected from non-stimulated animals are matured in vitro. Thirdly, in the human, fertilization can be performed with subfertile semen, whereas in the bovine, bulls selected for adequate semen quality are used. Fourthly, human embryos have been cultured routinely for 2 to 3 days (until the 16-32 cell stage, although culture for 7 days until the blastocyst stage has also been done), whereas bovine embryos are routinely cultured for 7 days until the morula or blastocyst stage. Lastly, the human recipient of the embryos is usually the donor of the oocytes, whereas the bovine embryo recipient and oocyte donor are two different animals. Whether these differences explain the observed discrepancy in direction of deviation (increased or decreased birth weight) of IVF offspring compared to AI or naturally conceived offspring is not known.

Theriogenology

577

Several epidemiological studies indicate that exposure of pregnant women to a sub-optimal environment can have severe effects on their children at birth and during later life. For instance, Barker and colleagues (20,22) have shown the effects of prenatal exposure of fetuses to the Dutch famine of 1944-1945. Food deprivation during pregnancy resulted in children with an increased risk of cardio-vascular problems (an increase blood pressure, related to reduced birth weight) and reduced glucose tolerance 50 years later. These observations are in line with the concept of "metabolic setting" from Hales and Barker (10) suggesting that poor maternal nutrition during pregnancy can lead to an adaptation of the fetus to this diminished nutrient supply and that this "programming" persists throughout life. It was shown that a normal birth weight is no guarantee for a normal metabolism and physiology. Conversely, a deviate birth weight may not always be indicative of a deviating metabolism and physiology. We hypothesize that the "metabolic setting" concept of Hales and Barker might also apply to the in vitro production of bovine (and human) embryos. The in vitro environment of bovine embryos is likely to be sub-optimal (unbalanced, an oversupply of certain nutrients and possibly lacking others) and effects of the IVP system are found at birth (large offspring syndrome and smaller children) and possibly later in life. What are the Causes

It is not yet clear what factor(s) cause(s) the "Large Offspring Syndrome" in sheep and cattle. Factors at any stage of the sequential process (maturation, fertilization, culture) may playa decisive role. Unlike the oviduct and uterine environments, the in vitro environment is generally a static system in which concentrations are fixed or changed only by metabolism of the cultured oocytes or embryos. Concentrations of components in the media are based on those found in the oviduct and uterus (29) or are known to support the growth of other types of cells. Culture media often are supplemented with proteins (serum, BSA, amino acids, etc) and layered on co-cultured cells, which secrete embryotrophic growth factors (buffalo rat liver (BRL) cells, oviduct epithelial cells, kidney epithelial cells, granulosa cells, etc) in an attempt to mimic in vivo circumstances (6). Obviously, these complex media are undefined (32). One of the factors most often mentioned in relation to the "Large Offspring Syndrome" is serum added to the culture medium. Serum contains numerous components (e.g. hormones) and as such provides a rich but undefined environment for embryo development (32). In addition, the production of embryotrophic growth factors by co-culture cells, the high oxygen tension in which embryos are cultured (20% vs 6-7% in the uterus), the static culture system and the high ammonium concentrations (waste product of protein metabolism) in the culture medium, are often mentioned as the cause of the unusually large offspring. Recently, from the perspective of the large offspring syndrome as well as from a quality control point of view, (semi-) defined culture media were developed (CR1aa (21); KSOM (3); SOF (29); GII2 medium (7». So far, most studies about birth weights of calves and lambs used Synthetic Oviduct Fluid (SOF), to which BSA and/or amino acids are added as an alternative protein source (SOFaaBSA) under low (5%) oxygen tension (see Table 1). Sheep Using SOF minus serum (SOF-) decreased birth weight and gestation length of lambs significantly compared with SOF plus serum (SOH (31); see Table 1). SOF- sheep embryos had

Theriogenology

578

fewer "lipid-like" inclusions, developed slower, and cell numbers were more similar to in vivo produced embryos (MOET) than SOH embryos (31). In addition, the allometric coefficients of heart, liver and kidneys ofSOF- lambs were similar to those of control lambs (MOET), whereas in SOF+ lambs allometric coefficients were equal to those of the co-culture+ group. These data are indicative of a more physiological development of-serum fetuses than +serum fetuses. Data from (16), however, indicate that SOF- still resulted in higher birth weights compared to control lambs (MOET). Table 1. Effect of the use of SOF with and without serum as culture medium on birth weight compared with different controls. Investigator

AI

MOET Co-cul+\ SOH 2

3

# animals species

15

sheep

21-39 11-13 5

sheep sheep sheep

c

5

cattle

c

5 11-16

cattle cattle

Thompson et al. (31) Holm et al. (12) Sinclair et al. (28) McEvoy et al. (16) McMillan et al. (17) Jacobsen et al. (13) Tricoire et al. (33)

SOF-

c c c

+4

c +

+

=

5

+

c

ICo-cuI: co-culture; 2c: control; 3_ : decreased birth weight compared to control; 4+ : increased

birth weight compared to control; 5=: same birth weight as control. Cattle Tricoire et al. (33) showed that although birth weight of calves from SOF+ and SOF- were not significantly different, SOF+ embryos showed a faster morphological development and had a significantly lower number of cells per embryo than SOF- embryos at corresponding developmental stages. Jacobsen et al. (13) did not observe any differences between AI, SOF+ and SOF- calves with respect to birth weight and gestation length. McMillan et al. (17) showed decreased fetal and placental weights of bovine SOF- vs AI pregnancies. The sometimes conflicting data with and without serum among studies might be explained by: 1) different batches of serum used (28); 2) the use of different sires in different groups; and 3) the often small numbers of animals per group. From the above studies it appears that, the use of SOF- tends to improve the quality ofIVP embryos (slower development, fewer lipid-like inclusions) and offspring (lower birth weight, more physiological allometric coefficients of organs) compared with culture media including serum and co-culture cells. However, in vitro maturation and fertilization may also influence embryo development and characteristics of offspring. Aims of this Study Aim 1. To accurately describe IVP offspring derived from a complex culture medium including serum and co-culture cells compared with control AI and MOET calves in large-scale field

579

Theriogenology

conditions. This IVP co-culture system is expected to result in the "Large Offspring Syndrome". For that purpose several parameters at birth (birth weight, gestation length, sex, calving ease, perinatal mortality, percentage male calves. etc) ofIVP co-culture calves were compared with MOET and AI calves born in the same time period in the same herds (Study I). Aim 2. To accurately describe IVP calves derived from a culture medium (SOF) without serum and co-culture cells in comparison with control AI and IVP co-culture calves. For that purpose, a study similar to Study I was conducted in field conditions where AI, IVP co-culture and IVP SOF calves were compared for the parameters mentioned above (Study 2a). In addition, physiological (blood oxygen saturation, heart and breathing frequency, etc) and behavioral parameters (vividness; reflexes, etc) were determined in semi-standardized conditions (one herd) ofIVP co-culture, IVP SOF and MOET calves (Study 2b). This way, the potential differences in physiology ofthe calves derived from different artificial reproduction techniques can be described in more detail. MATERIAL AND METHODS The work is presented in three parts: I) a comparison between co-culture IVP -, AI - and MOET calves born in field conditions; 2a) a comparison between co-culture IVP -, IVP SOF - and AI calves born in field conditions; 2b) a comparison between co-culture IVP -, IVP SOF - and MOET calves born in standardized semi-research conditions. Study I is an extension of a previously presented field study (14,36), i.e. MOET calves are included in the present study. Superovulation In Study 1 and 2b, superovulation was performed on first parity donor cows using FSH as previously described (23). Embryos were collected at Day 7 of the estrous cycle and frozen in 10% glycerol or ethylene glycol. Ovum Pick Up and In Vitro Production of Preimplantation Embryos Study 1. From January 1995 until 15 November 1997 ovwn pick-up (OPU) collections were made from 282 donors at 2 different locations throughout the Netherlands. Donors were 240 pregnant heifers and 42 first parity cows (Holstein Friesian [HF]) from the Delta open nucleus breeding program of Holland Genetics. Per animal, from pregnant heifers an average of 10.6 oocyte collections and from first parity cows an average of 42.0 collections were performed. Oocytes were collected twice a week, on Mondays and Thursdays. Collected cumulus oocyte complexes (COCs) were matured, fertilized and cultured as described previously (36). The culture system included 10% FCS and BRL co-culture cells. Transferable embryos (IETS Grade I and 2) were transported at 25°C in TCM199 to recipients at 267 participating farms throughout the Netherlands, including Holland Genetics recipient herds. When there were more embryos than recipients, blastocysts were frozen using conventional slow freezing in a 10% glycerol solution as previously described (37).

580

Theriogenology

Study 2. Between 15 November 1997 and 31 December 1998, OPU collections were made from 167 pregnant heifers (on average 9.8 collections per animal) and 19 first parity cows (on average 19.9 collections per animal). Starting 15 November 1997, presumptive zygotes on Day 1 were either cultured in the above mentioned co-culture system with 10% FCS or in a SOF culture system without serum nor co-cultured cells (108.5 mM NaCI, 7.2 mM KCI, 1.2 mM KH 2P04 , 0.74 mM MgS04 . 7H 20, 25 mM NaHC0 3, 1.8 mM CaCl 2.2Hp, 3.2 mM NaLactate, 0.33 mM NaPyruvate, essential and non-essential amino acids (Gibco, Life Technologies, Breda, The Netherlands) plus fatty-acid free, fraction V, BSA (Pentex, Bayer, Munchen, Gennany). In alternating weeks, presumptive zygotes on Day I (Day O=fertilization) developing from COCs colle~ted at one location were put in the co-culture system and zygotes developing from COCs from the other location were put in SOF or vice versa. Consequently, zygotes from donor cows of the 2 locations were randomized over the 2 culture systems. Embryo Transfer Study I and 2a. Fanners were solicited to participate in the field trials by an advertisement and were offered genetically valuable embryos produced from pregnant heifers and first parity cows at favorable financial conditions. Fanners were contracted to provide recipients that were healthy, fertile, regularly cycling cows rather than heifers because of the expected large calves. Recipients received an IVP embryo at Day 6, 7 or 8 during natural estrous cycles by a standard nonsurgical embryo transfer procedure. Recipients were rejected for obvious reasons only (e.g. endometritis) since most embryos were transferred fresh and numbers of embryos and recipients were matched. If there were surplus recipients, frozen-thawed IVP or MOET embryos (from super-stimulated heifers, Study 1) were transferred. In Study 1, data from the Holland Genetics recipient herds were included (embryos from first parity cows), whereas in Study 2 data collected on fanns outside Holland Genetics and data of the Holland Genetics recipient herd were separated in Study 2a and 2b, respectively. Study 2b. Embryos from first parity donor cows were transferred at the recipient herd of Holland Genetics (one location) under the same teons as mentioned above. Besides IVP co-culture and SOF embryos, MOET embryos were transferred in this herd. Control MOET calves were selected based on their pedigree. Those MOET calves with a pedigree that matched best to IVP co-culture and IVP SOF calves were included in Study 2b. Data Collection Study 1 and 2a. Pregnancies were confinned by palpation per rectum between Day 90 and 180 by a qualified veterinarian. Return to estrus was not registered accurately. Fanners were requested to register data on all IVP calves born and on 5 calves born prior to and 5 calves born after each IVP calf at the same fann, which served as controls. These controls consisted of mainly AI and some MOET calves. In Study 2a, AI calves born after I November 1997 at the same herds were also included, in order to increase numbers per herd. The following data were recorded by the fanners: calving date, birth weight (measured by a balance (Bio Enterprise, Vroomshoop, The Netherlands», sex, viability (alive, stillborn or dead within 24 h after birth), congenital malfonnations, ease of calving (classified in 6 categories: I = no assistance; 2 = some help; 3 = normal calving, help of I person; 4 = normal calving, help of2 persons; 5 = difficult calving; 6 = very difficult calving), and

Ther;ogenology

581

caesarian sections. In addition, data on gestation length (= calving date minus insemination date for AI calves and calving date minus date of start of estrus prior to embryo transfer for MOET and IVP calves), sex and perinatal mortality of MOET calves born on the contracted herds were included in Study 1, provided that the embryo was transplanted by Holland Genetics staff and that the calf was born in the period of interest. These data were retrieved from the national database. Study 2b. Estrus detection after transfer was performed 4 times a day by two experienced farm managers, resulting in reliable return data. Pregnant recipients at the Holland Genetics recipient herd were transported to a different location in the fifth month of pregnancy for calving. At and after calving, physiological, behavioral and endocrinological parameters were evaluated by one person, who was not aware ofthe origin ofthe calves (MOET, IVP co-culture or IVP SOF). In chronological order the following parameters were measured and recorded: time of start calving (i.e. time of first sight of membranes), time of delivery, cooperative behavior of the cow (good, moderate, bad) and clarity of the amniotic fluid (clear or not clear). Within 2 minutes after birth behavioral parameters were scored: color of mouth mucous membrane (pink, white, blue), use of breathing stimulus (yes/no; if the calfdid not lift its head within a minute, the farmer decided to use a straw to irritate the nose mucous), frequency ofbreathing (measured during 15 s) and interdigital painreflex (yes/no). Within 5 minutes after birth, heart pulse frequency and blood oxygen saturation were measured at the tail vein using a pulse-oxymeter (Instruvet, Amerongen, The Netherlands). This measurement took 2 to 15 minutes, depending on the accessibility of the tail vein. The general appearance of the calfwas scored as vivid or dull based on the reaction of the calf during measurement with the pulseoxymeter. Subsequently, birth weight, height and chest circumference were measured. Colostrum supplementation was started after these measurements. Each calf received the same amount of colostrum (a total of 400 gr of Col-O-Dan, freeze-dried, IBR-free powder; Damino, Gesten, Denmark) during the first 4 days after birth. Ultrasonography of Heart and Kidneys

In the first week after calving, ultrasound measurements were made of kidneys and heart of the calves born to investigate disproportionate organ development (organ dysmaturity). A 5 MHz sector probe of a Pie Medical 200V sector scanner (frame rate 22; Pie Medical, Maastricht, The Netherlands) was used to measure the diameter of the septum between left and right ventricle and the diameter of the left ventricle lumen and wall both in systole and diasystole. From these measurements, the Pie Medical software calculated the ejection fraction (the percentage of the total left ventricle volume that is ejected in one heartbeat). The whole procedure and measurement was done in duplicate. For ultrasonography of the kidneys, using Pie Medical software, an ellipse was drawn around the kidney on screen. The software subsequently calculated the surface of the kidney (in cm\ Measurements were done in triplicate. Data Management Field data on 5,353 AI calves, 1,123 MOET calves and 1,241lVP co-culture calves (Study 1) and on 1,764 AI-, 110 co-culture IVP, and 152 IVP SOF calves (Study 2a) were included. Gestation

Theriogenology

582

length, sex and perinatal mortality was recorded for all calves. The other parameters (birth weight, calving ease, congenital malformations and caesarian sections) were only available when farmers returned data. In Study 1, for 99% ofthe AI calves, 24% of the MOET calves and 78% ofthe IVP calves, at least one of the latterparan1eters was known. Ofthe 267 herds in Study 1,83% ofthe data sheets were returned by 221 herds, of which 130 herds returned all required data on IVP calves. Average birth weight ofIVP calves at herds returning a high percentage of data sheets (>67%) was the same as that at herds returning a low percentage of data sheets. In Study 2a, for 99% AI, 72% IVP co-culture and 66% IVP SOF calves, at least one ofthe parameters birth weight, calving ease, congenital malformations or caesarian sections, was known. Of the 128 herds in Study 2a, 82 herds returned 89% of the data sheets oflVP calves (of which 68 herds returned all required data sheets oflVP calves). At the Holland Genetics calving herds an additional 1,119 MOET calves and 211 IVP co-culture calves (Study I) and an additional 33 MOET -, 27 IVP co-culture- and 28 IVP SOF were born (Study 2b). From these animals all the above mentioned parameters are known. Table 2 shows some characteristics of recipients and inseminated cows that gave birth to the different categories of calves (AI, MOET or IVP). By including these parameters (except for fresh or frozen embryos) in the statistical model, differences in distribution of calves over these parameters were accounted for. Table 2. Characteristics of recipient/inseminated cows that gave birth to the different categories of calves (AI, MOET or IVP) in Study I, 2a and 2b. Parameter AI %HF recipient/dam Parity (%) 0 1 2 % fresh ET I

Study I MOET

IVP co-cull

89

65

76

29 23

69 16 15 25

48 21 32 85

48

AI

Study 2a Study 2b IVP IVP MOET IVP IVP co-cuI co-cuI SOF SOF 88 81 67 74 83 83 32 23 45

35 28 37 70

36 32 32 69

50 50 0 9

56

44 0 50

58 42 0 85

co-cuI: co-culture.

In Study 1, calves were sired by 1,066 different bulls, while IVP calves were sired by 72 bulls, 39 ofwhich had also both AI and MOET offspring. In all 267 herds at least 1 IVP calfwas produced, while 138 herds had both AI and MOET calves. In Study 2a, calves were sired by 431 bulls in 128 different herds and at least 1 IVP calf was born in each herd. In Study 2b, calves were sired by 28 bulls and were all born at the same farm. Congenital malformations, caesarian sections and male calves were evaluated as a percentage of all calves born. Subsequently, for the comparative analysis of birth weight, gestation length, perinatal mortality and ease of calving among IVP, AI and/or MOET calves (Studies 1 and 2a), 404 AI, 62 MOET and 78 IVP calves in Study I and 113 AI,3 IVP co-culture atld 3 IVP SOF calves in Study 2a were excluded from the database for the following reasons: I) aborted calves (stillborn prior to 260 days of gestation); 2) twins; or 3) calves with a congenital malformation. The final

583

Theriogenology

comparative data set of Study 1 consisted of 4,949 control AI -, 2,180 MOET - and 1,374 IVP calves. In Study 2a, 1,651 control AI, 1,07 IVP co-culture and 149 IVP SOF calves were included in the comparative analysis. Not all parameters were registered for all calves, so that grand totals may not always be the same. Birth weight, gestation length, perinatal mortality and ease of calving were compared using the MIXED procedure of the SAS statistical program (24) for Study 1 and 2a and the MODEL statement ofthe Genstat statistical program (9) for Study 2b. Data were analyzed using the following model: Yijklmnop

In which: gestation length, birth weight, ease of calving or percentage perinatal mortality

Yijklmnop

and all continuous parameters of Study 2b Fixed effects: Ti

type i of calf; i=AI, MOET or IVP; 3 levels

Sexj

sex j of calf; j=male or female; 2 levels

Pk

parity k of inseminated cow/recipient; k=O, I or> I; 3 levels

MJ

month of calving I; 1= Jan/Feb to NovlDec; 6 levels

Ym

year of calving m; m=1995 to 1998; 4 levels

Bn

breed n of inseminated cow/recipierit; n=Holstein Friesian, Maas-Rijn-Yssel, or other; 3 levels.

Random effects: herd 0 in which calf is born; 265 and 120 levels in Study 1 and 2a, respectively, sire p (sires with less than 4 offspring were grouped); 363 and 92 levels in Study 1 and 2a, respectively. All 2- and 3-way interactions of fixed effects and the interaction between sire and type of calf were included. Least square means (LSM) and standard errors (SE) were produced by a model including all significant main effects and interactions (P<0.05). In Study 2b, only the fixed effects were included in the statistical model. In addition, birth weight was included in the model for ultrasonographic kidney and heart data. Pregnancy rates and other bi- or multinomial parameters were analyzed using Chi-square analysis. RESULTS Ovum Pick Up, In Vitro Production and Embryo Transfer Study 1. An average of7.6 COCs were recovered per collection. Although no oocytes were found in 1.4% of the collections, COCs could be collected from all donor animals. At Day 4 of culture, an average 56% ofthe COCs was cleaved and by Day 7 of culture, an average of 17% had developed

Theriogen%gy

584

into transferable morulae or blastocysts (1.3 transferable embryo/collection). Of all collections, 44% resulted in zero transferable embryos and 17 animals (6%) had not produced any embryo after 1-13 collections. Pregnancy rates of fresh embryos were 42.4 (n=5,811) and of frozen embryos 42.4% (n=I,213). Study 2. An average of7.8 COCs were recovered per collection. Although no oocytes were found in 0.8% of the collections, COCs were collected from all donor animals. At Day 4 of culture, an average 59 and 61 % of the COCs were cleaved and by Day 7 of culture, an average of 14 and 17% had developed into transferable morulae or blastocysts (1.1 and 1.4 transferable embryo/collection) in the co-culture and SOF system, respectively. Of all collections, 45 and 39% ofthe collections did not result in a transferable embryo in the co-culture and SOF system, respectively. Four donor animals (2%) had not produced embryos after 1-10 collections.

In the co-culture and SOF systems, the percentages of morulae!bIastocysts produced were 33/67% (n=I,155) and 48/52% (n=I,240), respectively, indicating that embryos in SOF develop more slowly than in co-culture (P<0.05). Furthermore, 32% ofSOF morulae were Grade 1 (n=595) vs 26% of coculture morulae (n=381) (P<0.05). SOF morulae showed fewer lipid like inclusions (were lighter) and showed more compaction compared with co-culture morulae. Pregnancy rates of fresh and frozen co-culture and SOF embryos transferred from 15 November 1997 until March 1999 (Study 2a and 2b) on both contracted farms and the HG recipient herds are shown in Table 3. There were no significant differences between pregnancy rates of co-culture and SOF embryos (Grade 1, Grade 2, Grade 1&2; frozen and fresh; P>0.05; Chi-square analysis). Table 3. Pregnancy rates oflVP co-culture and SOF embryos. Co-culture

SOF

Fresh

Frozen

Fresh

Frozen

Grade 1

35.3 (245)3

42.1 (381)3

44.3 (432)3

38.8 (194)3

Grade 2

38.2(472)3

33.3(359)3

39.3(445)3

32.0(252)3

Total

37.2 (717)3

37.8 (740)3

41.8 (877)3

35.0 (446)3

3data with the same superscript in the same row are not significantly different (P>O.05; Chi-square analysis). Data on return to estrus after embryo transfer at the HG recipient herd are shown in Table 4. Pregnancy rates (100% - return%) of recipients receiving a MOET, IVP co-culture- or IVP SOF embryo were 45.6, 48.5 and 53.9%, respectively (P>0.05). In the MOET and IVP SOF group relatively more recipients returned to estrus before 31 days after estrus compared with recipients receiving an IVP co-culture embryo (PO.05).

585

Theriogenology Table 4. Return pattern of recipients after transfer of an MOET, IVP co-culture or IVP SOF embryos at the HG recipient herd. Parameter

IVP co-culture (n=157) 85(51.5)3 3 68.2 b 19.0

IVP SOF (n=101) 47(46.1)3 3 80.9

11-18 d2

MOET (n=465) 253(54.4)3 3 80.6 3 10.8

19-23 d2

72.0

50.0

73.7

24-31 d2

Total Return(%) %return 0-31 d

1

13.2 a

17.2

31.0

13.2

% return 32-52 d

1

13.1

20.0

10.6

% return 53-73 d

1

5.1

8.3

8.5

% return> 73 d

l

1.2

3.5

0.0

) Calculated as percentage of total return. No significant difference in distribution of returns over the 4 classes (0-31 d, 32-52 d, 53-73 d and> 73 d) among the 3 groups. Difference in distribution of return over 2 classes (0-31 d and> 31 d) among the 3 groups is statistically significant (P=0.05; Chi-square analysis). Calculated as percentage of return 0-31 d. Difference in distribution of return over the 3 classes is statistically significant (P<0.05; Chi-square analysis) among the 3 groups.

2

ab data with different superscripts in the same row are significantly different (P<0.05; Chi-square analysis). Characteristics of Calves Born Study. I and 2a. Percentage abortions, caesanan sections, male calves and congenital malformations are shown in Table 5. Table 5. Percentage abortions, caesarian sections (C-sections), male calves and calves with a congenital malformation in Study 1 and 2a. Parameter AI % Abortions

Study 1 MOET

IVP CO-CUll

AI

Study 2a IVP co-cuI

IVP SOF

1.3(5,353)2a

1.1 (2,242) a

2.6( I,452) b

0.5(1,764) a

0(110)a

1.3(152) a

% Congenital 0.8(5,353) a malformation

1.5(1,089) b

3.7(1,129)c

0.6(1,764) a

3.7(81) b

1.0(97)3b

%C-sections

8.4(1,107)b

11.2(1,179)c

0.7(1,764) a

3.8(80)b

8.3(96)b

% Male

1.5(3,313)a

49.8(5,353)a 53.7(2,194)b 52.9(1,415)b 52.8(1,764)a 56.1(110) a 54.9(152)3

3b data with a different superscript in the same row within each study are significantly different (P<0.05; Chi-square analysis). I co-cuI: co-culture; 2 numbers between brackets.

Theriogenology

586

0

~

LO

N V

N

N

I'-

I'-

0

I

I'-

co I'-C\I

I'-I'--

N

co

I'-

N

OJ I'--

N

0

co

C\I

....co N

C\I

('I)

N

N

co

co

~

N

LO

co

N

co co C\I

I'--

co

N

~

N

OJ

co

N

0 OJ

N

0

0)

N /I

I'-

N

Geslalill1length (days) Figure 1. LSM and SE for birth weight of AI, ET and IVP co-culture calves for different gestation lengths (Study I). In Study I, the percentage of aborted IVP fetuses was twice that of AI and MOET fetuses (Po.05). The percentage of caesarian sections was significantly higher in MOET and IVP calves than in AI calves in both studies (p
587

Theriogenology

MOET calves vs IVP calves increased with longer gestation (P<0.05). In addition to a higher average, the standard deviation of birth weight ofIVP co-culture calves was also higher (standard deviation of 5.3,5.0 and 7.1 kg for AI, MOET and IVP co-culture calves, respectively). Both MOET and IVP calves had significantly longer gestation periods than AI calves (P<0.001). Gestation length ofMOET calves was significantly shorter than ofIVP calves (P<0.05). The range among different herds in gestation length was 5 d (P<0.05), the range among different sires was 9 d (P<0.05). IVP calves had a 1.7% (P<0.06) and 2.9% (P
Parameter

AI

Birth weight (kg)

42.7 ± 0.2 a

Gestation length (d)

281.2 ± 0.2 a (4,946)

Perinatal mortality (%) .

Easeofcalvmg

5.3 ± 0.4

ab

(4,878)

(4,949)

MOET

(n)

43.4 ± 0.3 a

(1,058)

282.1 ± 0.2 b (2,139) 4.6 ± 0.7

a

l a b

2.4±0.04

(4,861)

2.7 ± 0.07

IVP co-culture (n) 47.1 ±O.3

b

(1,049)

283.6 ± 0.2 C (1,358)

(2,180)

7.5 ± 0.7 b

(991)

3.2±0.05

(1,374) c

(971)

in 6 different classes (I = easy, to 6 = very difficult). a,b,c data with a different superscript in the same row are significantly different (P<0.05). 1 ease of calving was scored

The process of calving was significantly heavier for both MOET and IVP compared with AI calves (P
AI

IVP co-culture (n)

IVP SOF

(n)

42.3 ± 0.3 a (1,604)

46.0 ± 0.6 b

(77)

46.1 ± 0.6 b

(93)

280.4 ± 0.3 a (1,648)

282.6±0.7

(104)

281.8 ± 0.6

Perinatal mortality (%) 4.7 ± 0.6 a Ease of calving

l

(n)

(1,651)

2.29 ± 0.06 a (1,620)

b

7.3 ± 2.1 a 3.00 ± 0.14 b

(107) (76)

5.2± 1.8

ab

a

2.82 ± 0.13 b

1 ease of calving was scored in 6 different classes. a,b,c

data with a different superscript in the same row are significantly different (P<0.05).

(141) (149) (89)

Theriogenology

588

In Study 2a, both IVP co-culture and IVP SOF calves were significantly heavier and had more difficult births than AI calves (P<0.05). IVP SOF calves had non-significantly longer gestations, whereas IVP co-culture calves had significantly longer gestations than Al calves. Although the difference between SOF and co-culture IVP calves was not significant for perinatal mortality and ease of calving, SOF calves showed values intennediate between AI and IVP co-culture calves. Results of AI and IVP co-culture calves in Study 2a were similar to results of AI and IVP co-culture calves in Study 1. Table 8 shows LSM and SE of birth weight for the interaction between type of calf and sex of Study 2a (P<0.05). While for IVP co-culture and AI calves the difference between males and females was 1.6 and 2.8 kg, this was 5.6 kg for IVP SOF calves (P<0.05), indicating that SOF females tended towards lower birth weights than co-culture females (P=O.08), whereas for male calves this effect was reversed (P<0.05). Table 8. LSM and SE of birth weight for the interaction between type of calf and sex (number of observations). AI Sex

(n) a

Male

43.7±OJ

Female

40.9 ± OJ a (754)

(850)

IVP co-culture b 46.8±0.8

(n)

IVP SOF

(n)

(38)

49.0±0.7

45.2 ± 0.8 b

(39)

43.2 ± 0.8 b (42)

c

(51)

a,b,c data with a different superscript in the same row are significantly different (P<0.05). Study 2b. A total of34 MOET, 32 IVP co-culture and 33 IVP SOF calves were born at the HG calving herd from 15 August 1998 until 11 July 1999. Percentage abortions, caesarian sections, male calves and congenital malfonnations, are shown in Table 9. LSM and SE ofbirth weight, gestation length and ease of calving are shown in Table 10. Table 11 shows LSM and SE of different physiological and behavioral parameters ofMOET, IVP co-culture and IVP SOF calves. Table 9. Percentage of abortions, perinatal mortality, caesarian sections (C-sections), stillborn, male calves and calves with a congenital malformation of Study 2b. Parameter

MOET (n'=34)

% Abortions

IVP co-culture (n=32)

IVP SOF (n=33)

3.1 a (l hydro-allantois)

% Perinatal mortality

3.1 a

% Congenital malformation % C-sections

3. I a 2.9 a

25.0 b O a

% Male

35.7 a

54.8 a

48.4 a

ab data with the same superscript in the same row do are not significantly different (P>0.05; Chisquare analysis).

589

Theriogenology

Percentage abortions, perinatal mortality and congenital malformations, based on low numbers of observations, did not show large differences between the 3 groups (P>0.05). Percentage male calves of the MOET group was quite low, but not significantly different from IVP co-culture and IVP SOF calves (P>0.05; Chi-square analysis). The percentage of caesarian sections was significantly higher in IVP co-culture calves than in MOET and IVP SOF calves (P<0.05). IVP SOF calves had a significantly lower birth weight than IVP co-culture calves (P<0.05) and had no significantly different birth weight from MOET calves. The birth weight difference between male and female calves was 3.0, 2.1 and 0.6 for MOET, IVP co-culture and SOF, respectively (P>0.05). Gestation length was similar for the 3 groups. Ease of calving of IVP SOF calves was intermediate to that of MOET and NP co-culture calves. Birth weight and gestation length ofMOET and IVP co-culture calves showed lower values compared with Study I and 2a in this one herd. LSM and SE of different parameters per type of calf (number of observations) of Study 2b.

Table 10.

Parameter

MOET

Birth weight (kg) Gestation length (d) .

Ease of calvmg I

I

(n)

IVP co-culture (n)

IVP SOF

(n)

41.3 ± 1.0 (30) a

46.4 ± 1.0

(30) b

42.3 ± 1.0 (30) a

279.9 ± 1.3 (30) a

279.2 ± 1.3

(30) a

279.6 ± 1.2 (30) a

2.9 ± 0.18 (29)

a b c

4.0 ± 0.20

(22)

3.4 ± 0.17 (30)

ease of calving was scored in 6 different classes. data with a different superscript in the same row are significantly different (P<0.05).

a,b,c

Of the IVP co-culture calves, 8 were born by caesarian section. If there were any differences between these calves and co-culture calves born naturally, this is mentioned in the text. For all calves, the amniotic fluid was clear. The parameters color of mouth mucous and interdigital painreflex did not show any differences among the groups. IVP co-culture calves were less vivid after birth than NP SOF and MOET calves and, especially after a caesarian, significantly more often required a breathing stimulus compared with MOET calves (PO.05). The oxygen saturation level was significantly higher in IVP co-culture and SOF calves compared with MOET calves. Heart pulse frequency was significantly lower in the IVP co-culture group, which is partly caused by the very low heart pulse frequency of the caesarian section animals in this group (124 and 141 beats/min for caesarian section and normal calves, respectively). Height and chest circumference ofSOF calves was intermediate between MOET and IVP co-culture calves, analogous to birth weight. Kidney surface of MOET calves was larger compared with IVP calves (P>0.05). The average coefficient of variation (CV) of the triplicate kidney measurements was 11.8%. The intra-ventricular septum (IVS) during diastole was significantly thicker for IVP compared with MOET calves (P<0.05). There were no significant differences among the 3 groups for left ventricle lumen (LV) during diastole, whereas the left ventricle wall (LVW) during diastole was significantly thicker for IVP co-culture calves compared with MOET and NP SOF calves (P<0.05). The average CV of duplicate heart measurements were 10.8,8.6, 12.2 and 2.3% for diastole (D) IVS, LV and LVW, and ejection fraction, respectively. Data

Theriogenology

590

during systole showed the same tendency, but were not significantly different among the 3 groups (P>O.05). Including birth weight in the statistical model, showed a significant effect of birth weight onD-IVS and D-LVW. The significant difference in D-LVW became non-significant after inclusion of birth weight in the statistical model, but the ranking among the 3 groups remained the same. CONCLUSIONS The presented results provide evidence for the following conclusions: 1) Both IVP co-culture and MOET calves have higher birth weights, longer gestations, a higher percentage of male calves, congenital malformations, caesarian sections and perinatal mortality and a more difficult calving process compared with AI calves (Study I). MOET calves show intermediate values between AI and IVP calves and are not always significantly different from AI calves (Tables 5 and 6). 2) Culturing in vitro-produced zygotes in a semi-defined medium, SOF, instead of a complex coculture plus serum system, resulted in slower developing embryos (a higher percentage of morulae relative to blastocysts at Day 7 of culture) and morulae of a higher quality (reflected in different morphology). In field conditions however, SOF did not result in significant differences between calves and calving characteristics compared with co-culture calves, although gestation length, perinatal mortality, percentage of congenital malformations and ease of calving ofSOF calves were intermediate to MOET and IVP co-culture calves. Birth weight of SOF females tended to be lower than that of co-culture females (P==O.08), SOF males were significantly heavier than co-culture males (P
591

Theriogenology Table 11. Characteristics of different types of live born calves and of the calving of Study 2b.

MOET (n=30)

IVP co-culture (n=30)

IVP SOF (n=30)

22 0 0 8

18 3 0 9

24 1 1 4

b

ab

7

15 14 I

8 18 4

19 2 9

20 9 1

22

Calving process (min)

61.8±8.5(19)a

64.0±8.0(21)a

76.1±7.3(25)a

Breathing frequency

41.7 ± 1.5 (20) a

42.8 ± 1.2 (29) a

43.9 ± 1.3 (25) a

Heart pulse frequency

156 ± 8.2 (20) a

128 ± 6.7 (29) b

152 ± 7.1 (25) a

Oxygen saturation

87.7 ± 1.7 (20) a

92.5 ± 1.4 (28) b

92.2 ± 1.5 (25) b

Height (em)

79.6 ± 0.7 (23) a

81.6 ± 0.6 (28) b

80.3 ± 0.7 (25) ab

77.1 ± 0.8 (23)

80.5 ± 0.7 (29) b

77.6 ± 0.7 (25) a

28.9 ± 1.9 (19) a

28.7 ± 1.7 (26) a

Category/Characteristic

Cooperation cow Good Moderate Bad Unknown Use of breathing stimulus Yes No Unknown General appearance Vivid Dull Unknown

Chest circumference (em) Kidney surface (cm

2 )

5 18

a

31.4 ± 1.9 (21) a

3 5

D-Intra-ventricular septum 1

10.5 ± 0.3 (24) a

12.0 ± 0.3 (23) b

11.8 ± 0.3 (26) b

D-Left ventricle lumen

33.2 ± 0.8 (24) a

31.2 ± 0.8 (23) a

31.6 ± 0.8 (26) a

D-Left ventricle wall

11.8 ± 0.6 (24) a

14.1 ± 0.5 (23) b

12.4 ± 0.5 (26) a

Ejection fraction (%)

88.0 ± 1.2 (24) a

88.9 ± 1.2 (23) a

87.2 ± 1.2 (26) a

a,b,c data with a different superscript in the same row are significantly different (P<0.05). 1 D: diastole. In MOET, maturation is altered by administration of superovulatory honnones. Vos (35) has shown that approximately 50% of the oocytes that ovulate upon superovulation can be considered to be "nonnal" based on their morphology and estrogen and progesterone levels in the follicular fluid. Superovulated, in vivo matured oocytes, when fertilized and cultured in vitro, develop into blastocysts at a higher rate than in vitro matured oocytes (50.3% vs 29.8% at Day 9; 34). During and following superovulation, non-physiological numbers of ovulatory follicles and corpora lutea

592

Theriogenology

produce estrogen and progesterone, respectively, which alter the local environment in oviduct and uterine. These changes might affect sperm transport, maturation and selection in such a way that fertilization becomes less effective and efficient. Higher local progesterone levels are thought to induce faster development of the embryos in superovulated compared with non-superovulated cows. During in vitro production of embryos, not only the maturation step is altered dramatically (immature oocytes are collected from the ovary in different stages of atresia or growth), but also fertilization and culture are performed in a way far removed from the natural processes. The fact that these steps take place outside the female tract will have an effect on both the quantity and the quality of embryos and offspring produced. It seems that in the current MOET and IVP procedures we have surpassed the limits of mother nature's flexibility. In field Study I, IVP co-culture calves bom in 3 subsequent years were compared with AI and MOET calves, born in the same herds in the same time period. Although numbers in the present study are larger compared with data previously shown (36), the same conclusion holds, specifically that IVP co-culture calves are significantly different from AI calves with regard to birth weight, gestation length, perinatal mortality, ease of calving, percentage of congenital malformations, male calves and abortions. In the present experiment, data on MOET calves were included so that they could be compared with AI and IVP co-culture calving data. The data show that MOET calves also deviate significantly from AI calves with respect to the above mentioned parameters. Wilson et al. (40) found that MOET calves were approximately 8% heavier at birth than AI calves. Sinclair et aI. (26) showed earlier that gestation length of MOET calves is longer than that of AI calves. It is presently unclear which phase of in vivo embryo production is responsible for the deviations ofthe MOET calves born, but oocyte maturation seems to play an important role and requires more fundamental research. In field Study 2a, with relatively low numbers of observations for the IVP calves, no significant differences could be detected between SOF and co-culture IVP calves in. birth weight, gestation length, perinatal mortality, ease of calving and percentage of male calves and congenital malformations. However, except for birth weight, IVP SOF calves showed values intermediate between AI and IVP co-culture calves. As shown in Study I, factors like herd, sire, parity, etc significantly affect birth weight, gestation length, perinatal mortality and ease of calving. In field studies these factors need to be randomized over the different treatment groups investigated and numbers need to be high enough in order to rule out chance. Therefore, data need to be interpreted with caution. The extremely high weight for SOF males remains unexplained. The percentage returned data sheets of IVP calves in Study I and 2a is indicative of no selection of returned data. Recipients that received an MOET or IVP SOF embryo tended to return to estrus more regularly than recipients receiving an IVP co-culture embryo (Study 2b). McMillan et a!. (17) have shown that IVP SOF embryos develop smaller and functionally inadequate membranes (allantois), responsible for lower pregnancy rates and more irregular returns to estrus than AI embryos. We show that in semi-standardized conditions (Study 2b), IVP SOF embryos do not only result in a higher pregnancy rate in comparison with MOET (!) and IVP co-culture embryos, but also produce a return pattern and calf characteristics that are more corresponding to those of MOET embryos than to the results of IVP co-culture embryos.

Theriogenology

593

In Study 2b, SOF calves showed a significantly lower birth weight, had shorter gestations, easier calvings, and more normal in parameters like body measurements than IVP co-culture calves and were not significantly different from MOET calves in these respects. As shown in Study 1, MOET calves are not a golden reference, but since they were the only other type of calves available at the HG herd, they served as the reference group in this study. The fact that in Study 2b results were statistically significantly different may be explained by the fact that in Study 2b, circumstances were more controlled (the factors herd was excluded). Again, because of the relatively low number of observations, the interpretation of data should be done with caution. In Study 1 and 2b, the percentage of frozen embryos was higher in MOET than IVP calves. In IVP, only the most advanced, highest grade embryos (Grade 1 (expanded) blastocysts) are frozen, since pregnancy rates of these embryos are highest, whereas MOET embryos are frozen based on their grade only. In earlier analyses of IVP calves (18) we have shown that freezing seems to increase birth weight of IVP offspring (calves derived from frozen embryos were 1 kg heavier than those derived from fresh embryos). Whether the selection of embryos is responsible for these observations is not known. Assuming that the effect of freezing on birth weight is similar in MOET calves, we tend to underestimate the effect of IVP on birth weight in the present study. Additional measurements on behavior and physiology in Study 2b show that lVP calves not only deviated from MOET calves with respect to birth weight, gestation length, perinatal mortality etc, but also with respect to oxygen saturation (both IVP SOF and co-culture calves showed higher values compared with MOET calves), heart beat frequency and some measures of the heart. Coculture calves, especially born by caesarian section, have a significantly reduced heart beat frequency compared with MOET and IVP SOF calves. The septum and left ventricular wall of lVP co-culture and/or SOF calves was significantly thicker compared with MOET calves, indicative of heart dysmaturity. Those results confirm earlier data from Sinclairs group (27). Concept for Explaining Effects oflVP We propose that during in vitro culture of bovine zygotes, the "metabolic setting" concept of Hales and Barker (10) applies. An imbalance of nutrients (a relatively rich culture medium, serum, and growth factors produced by co-culture cells and possibly lacking others nutrients) may result in the relatively fast development of morphologically and functionally deviate embryos and fetuses and large calves compared with AI calves. Further, compounds may be added to the IVP media which cause deviations in development by their estrogenic action such as phenol-red. This metabolic setting may be the sum of 1) disturbances in oocyte maturation; 2) disturbances in gene regulation and expressions; 3) alterations in cell kinetics, i.e. accelerated growth and inadequate compaction; and/or 4) aberrations in the energy metabolism ofthe embryo. These aberrations occurring during early embryonic development may have their effect at birth and perhaps later in life. Effects of fVP on Reproduction Later in Life Routinely, based on their expected genetic value, 20-25% ofthe IVP calves born from our IVP field trials are taken back into our open nucleus program (HF) to be reared and subsequently tested together with animals born after AI or MOET. When proven to be genetically superior, they are selected to produce the next generation and their genes are disseminated into the population. Bulls

Theriogenology

594

start producing semen at one year of age and their semen is used to enable accurate estimation of their breeding value. Heifers are reared, superovulated once or twice from 13 months of age, thereafter inseminated during a normal cycle at approximately 17 months of age, and OPU and IVP are performed during the first three months of subsequent pregnancy. Upon calving, milk production is evaluated in standardized circumstances at one of two test herds of Holland Genetics. By now, we have performed preliminary analyses (relatively low numbers of animals) on semen production data and insemination results (non return rates) of IVP co-culture bulls and superovulation-, AI- and OPU/IVP results of IVP co-culture heifers in comparison with AI and MOET animals that produced semen or embryos in the same time period. Data were analyzed using analysis of variance, taking into account factors explaining both variance within and between bulls or heifers for the different a parameters. Tables 12 and 13 show a summary of LSM and SE . Table 12. Semen production data oflVP co-culture bulls compared with AI and MOET bulls at 1 year of age. Parameter Volume ofraw ejaculate (ml) Concentration (cells/ml) I Output (no. of produced cells) )

% motile cells prior to freezing .

2

2

AI 3.9 ± 0.1 a

MOET 3.9 ± 0.1 a

1,060 ± 31 ab

1,016 ± 16 a a 3,920 ± 77

4,387 ± 203 b

71.6 ± 0.5 a

71.6 ± 0.2 a

69.5 ± 0.6 b

a

a

a

4,045 ± 144 ab

IVP co-culture 3.8 ± 0.1 a 1,159 ± 43 b

% motile cells post-thaw 42.1 ± 0.5 42.7 ± 0.3 42.1 ± 0.7 Number of observations: AI: 1,123 ejaculates from 66 bulls; MOET: 4,166 ejaculates from 218 bulls; IVP: 589 ejaculates from 39 bulls. 6 1 values x 10 2 estimated using a phase contrast microscope at 200 x magnification. ab data with a different superscript in the same row are significantly different (P<0.05). Table 12 indicates that IVP co-culture bulls have a significantly higher semen concentration and semen output and a significantly lower percentage motile cells prior to freezing (P<0.05) compared with AI and MOET bulls. Non return rates of AI, MOET and IVP co-eulture bulls were 68.9 (range 63.8 to 72.7), 68.7 (range 56.3 to 73.9) and 68.4 (range 59.3 to 74.0), based on approximately 850 inseminations from each of 66, 218 and 39 AI, MOET and IVP bulls, respectively (P>0.05). Table 13 shows no significant differences among AI, MOET and IVP co-culture heifers in superovulation-, AI nor OPU/IVP results. Considering the size of the significant effects, the low number of observations, the fact that only part of all calves born from our IVP field trials are taken back into the open nucleus program and that again only part of these calves are actually evaluated (selection based on genetics takes place during the rearing period), these results should be interpreted with caution. However, so far, no indications of a negative effect oflVP on semen production and non return rates of IVP bulls nor reproduction results of IVP heifers are observed.

aunpublished observations.

Theriogenology

595

Table 13. Superovulation, AI and OPU/IVP results ofIVP heifers compared with AI and MOET heifers at 1 year of age. Procedure

Parameter

Superovulation 1 Total # of embryos # vital embryos % vital embryos 4 2 # insernlpregn AI OPU/IVP 3

# COCs

# embryos at Day 7 % embryos at Day 7

AI

MOET

IVP

5.5 ± 0.9 3.6 ± 0.7 61 ± 5

5.6 ± 0.6 3.8 ± 0.5 66 ± 4

4.8 ± 0.8 3.2 ± 0.6 64 ± 5

1.7 ± 0.2

1.6 ± 0.1

1.7 ± 0.1

7.0 ± 2.6 1.1 ± 0.2 16.4 ± 2.1

8.6 ± 2.5 1.2 ± 0.1 15.4 ± 0.9

8.4 ± 2.5 1.0 ± 0.2 13.2 ± 1.6

1 Superovulation: 105 collections from 56 AI heifers; 782 collections from 420 MOET heifers; 218 collections from 128 IVP heifers; 2 AI: 32 inseminations of20 AI; 181 OF 117 MOET and 92 of 56 IVP; not all data available at time of analysis; 3 OPUIIVP: 236 OPU collections of23 AI, 1,753-Bf 165 MOET and 446 of 49 IVP pregnant heifers. 4inseminated over pregnant.

Improving culture conditions by using a medium in which serum is substituted by BSA and amino acids and co-culture cells are deleted may normalize embryo development and characteristics of calves born. We will continue to follow IVP co-culture and IVP SOF calves later in life and to optimize both maturation and culture conditions with respect to embryo formation rates and the quality of calves born. REFERENCES 1. Behboodi E, Anderson GB, BonDurant RH, Cagill SL, Kreuscher BR, Medrano JF, Murray ill. Birth of large calves that developed from in vitro-derived embryos. Theriogenology 1995; 44:227-232. 2. Edwards RG, Steptoe PC. Birth after the preimplantation of a human embryo. Lancet 1978; 2:366. 3. Erbach GT, Lawitts JA, Papaioannou VE, Biggers JD. Differential growth of the mouse preimplantation embryo in chemically defined media. BioI Reprod 1994;50:1027-1033. 4. Farin PW, Farin CEo Transfer ofbovin embryos produced in vivo or in vitro: survival and fetal development. BioI Reprod 1995;52:676-682. 5. Fukada YM, Ichikawa M, Naito K, Toyoda Y. Birth of normal calves resulting from bovine oocytes matured, fertilized and cultured with cumulus cells in vitro up to the blastocyst stage BioI Reprod 1990;42: 114-119. 6. Gandolfi F, Moor RM. Stimulation of early embryonic development in the sheep by co-culture with oviduct epithelial cells. J Reprod FertiI1987;81:23-28. 7. Gardner DK Lane M. Culture of viable human blastocysts in defined sequential serum-free media. Hum Reprod. 1998;13:101-112.

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