Dissertation (PhD(Agric))--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: This study is a search for a quantifiable measure which estimates the genetic merit of female animals’ breeding efficiency. For practical reasons, such a measure must be both simple and inexpensive to record, irrespective of the herd management strategy. A literature investigation was undertaken to summarize breeding objectives for reproduction efficiency and to review different ways of expressing genetic reproduction efficiency. Traits to assess these in terms of the breeding objective, merits and requirements in terms of data collection are discussed. During the lifetime of a cow events occur which influence her fertility. A distinction is made between component traits and aggregate traits: a component trait points to one event, while aggregate traits are composites of more than one event. Although all the traits discussed seem relevant for breeding value estimation, the practical application depends on the herd management system in use. Age at first calving and days to calving are component traits that are easily and inexpensively measurable. Heritability estimates for the age at first calving were moderate. The heritability estimated for days to calving was 0.09. Calving rate comes close to the overall breeding objective. The estimated heritability of calving rate is low (0.04), resulting in slow genetic improvement. Calving success was defined and investigated even though some constraints exist. A sire model proved that genetic variation exists for calving success on the underlying scale. The corresponding heritability estimate was 0.27. Three categorical traits were defined. For stayability a sire variance of 0.41 was estimated with a heritabitity on the underlying scale of 0.27. The sire variances and heritabilities estimated for retention and calf tempo were high. Of the three traits, calf tempo is the one that reflects the true fertility of the bull’s female progeny. Calf tempo was redefined as net breeding merit, a trait describing the retention of male animals and the reproductive performance of their female offspring. The obtained sire variances show that the trait is heritable and can be improved by selection. Net breeding merit gives an indication of the ‘success’ of sires in a given population. A heritability estimate of 0.20 was estimated on a data set comprising offspring of bulls older than nine years, but when offspring of all sires were included, heritability estimates of 0.08 and 0.11 for the Afrikaner and Bonsmara, respectively, were found. Adjusting for young females was investigated by using the best linear unbiased estimate (BLUE) deviations to derive adjustment factors for herd level in order to predict performance for net breeding merit. Variation in the BLUE deviations occurred between all age class groups for the Afrikaner, whereas for the Bonsmara the variation between the BLUE deviations for the 3 year olds seems greater than the variation in the other age groups. It is suggested that the standardized curve for herd performance level derived from the BLUE deviations be used to adjust the phenotypic values of younger animals. This way the comparison between older and younger animals should be more valid. In conclusion, reproductive traits are heritable and genetic improvement can be achieved through selection. Any economical viable beef enterprise should include at least one trait in their selection criteria that will improve the reproductive efficiency. / AFRIKAANSE OPSOMMING: Hierdie studie is ‘n ondersoek na ‘n kwantifiseerbare maatstaf wat die genetiese meriete van vroulike diere se teeldoeltreffendheid beraam. Om praktiese redes moet so ‘n beraming sowel eenvoudig as goedkoop wees om te bepaal, onafhanklik van die kudde bestuurstrategie. ‘n Literatuurstudie is onderneem om die teeldoeleindes vir reproduktiewe doeltreffendheid op te som, sowel as om die verskillende wyses van genetiese reproduksiedoeltreffendheid beskrywing onder oë te neem. Verskeie eienskappe om hierdie beskrywings in terme van teeldoeleindes, meriete en dataversamelings-vereistes te raam, word bespreek. Gedurende ‘n koei se leeftyd kom gebeurtenisse voor wat haar vrugbaarheid beïnvloed. Daar word onderskei tussen komponenteienskappe en aggregaateienskappe: ‘n komponenteienskap verwys na een gebeurtenis, terwyl aggregaateienskappe na samestellings van meer as een gebeurtenis verwys. Hoewel al die eienskappe wat bespreek word relevant voorkom, sal die praktiese toepassing afhang van die kuddebestuurstelsel in gebruik. Ouderdom by eerste kalwing en dae tot kalwing is komponenteienskappe wat maklik en goedkoop bepaal kan word. Oorerflikheidsramings vir die ouderdom van eerste kalwing was matig. Die oorerflikheidsraming vir dae tot kalwing was 0.09. Kalffrekwensie is baie na aan die oorkoepelende teeldoelwit. Die geraamde oorerflikheid vir kalffrekwensie is laag (0.04), wat stadige genetiese verbetering tot gevolg het. Kalfsukses is gedefinieer en ondersoek, hoewel enkele beperkings bestaan het. ‘n Vaar-model het aangetoon dat genetiese variasie ten opsigte van kalfsukses op die onderliggende skaal bestaan. Die ooreenkomstige oorerflikheidsraming was 0.27. Drie kategoriese eienskappe is gedefinieer. Vir blyvermoë in die kudde is ‘n vaar-variansie van 0.41 geraam, met ‘n oorerflikheid van 0.27 op die onderliggende skaal. Die vaar-variansies en oorerflikhede wat vir retensie en kalftempo bereken is, was hoog. Van die drie eienskappe is kalftempo die een wat die ware vrugbaarheid van die bul se vroulike nageslag reflekteer. Kalftempo is herdefinieer as netto teelmeriete, ‘n eienskap wat die retensie van manlike diere en die reproduktiewe prestasie van hulle vroulike nasate beskryf. Die verkreë vaar-variasies wys dat die eienskap oorerflik is en verbeter kan word met seleksie. Netto teelmeriete gee ‘n aanduiding van die “sukses” van ‘n vaar in ‘n gegewe populasie. ‘n Oorerflikheidsraming van 0.30 is verkry op ‘n datastel bestaande uit die nageslag van bulle ouer as nege jaar, maar as die nageslag van alle vaars ingesluit is, was die oorerflikheidsraming onderskeidelik 0.08 en 0.11 vir die Afrikaner en Bonsmara. Aanpassing vir jong vroulike diere is ondersoek deur gebruik te maak van die beste lineêre onpartydige beramings (BLUE) om korreksiefaktore vir die kuddevlak te verkry, ten einde die prestasie ten opsigte van netto teelmeriete te voorspel. Variasies in die BLUE afwykings het voorgekom tussen alle ouderdomsgroepe vir die Afrikaner, terwyl vir die Bonsmara die variasie tussen BLUE afwykings vir die 3-jaar oud diere groter was as vir die ander ouderdomsgroepe. Dit word voorgestel dat die gestandardiseerde kurwe vir kuddeprestasievlak wat afgelei word van BLUE afwykings gebruik word om die fenotipiesewaardes van jonger diere aan te pas. Op hierdie wyse behoort die vergelyking tussen ouer en jonger diere meer geldig te wees. Ten slotte, reproduktiewe eienskappe is oorerfbaar en genetiese vordering is moontlik deur seleksie. Enige ekonomies lewensvatbare vleisbees-onderneming behoort ten minste een eienskap wat die reproduktiewe doeltreffendhied sal verbeter, in te sluit in seleksie kriteria.

Four experiments were conducted to investigate methods of producing transgenic bovine embryos entirely in vitro. Experiment 1 examined the effect of DNA microinjection at 11, 15 and 19 h after fertilization (haf) on survival rate and DNA detection frequency by polymerase chain reaction (PCR). There was no difference in transgene detection frequency between treatments (53% at 11; 50% at 15; 48% at 19 haf). Of all injected embryos developing to the morula or blastocyst stage after 7 d in culture, 89% tested positive for the presence of the transgene by PCR. Greater developmental efficiencies can be obtained when injection is performed early in pronuclear formation (7% (11/161) at 11; 4% (61159) at 15; 1 % (1/165) at 19 haf; p<0.05). Experiment 2 examined the effect of microinjection of DNA into the germinal vesicle (gv) of bovine oocytes on subsequent development and detection of the transgene. Injection of the transgene into the gv reduced developmental rates compared to controls (control=23% (89/384); non-injected=9% (23/250); GV injected=5% (12/259); p<O.05). Transgene detection frequency was 64% (37/58). Injection of bovine oocytes before fertilization results in viable embryos containing the transgene, although at low frequencies. Experiment 3 was designed to examine whether the frequency of microinjected DNA detection by peR In whole bovine embryos would decline over a 21 d culture period. At d 0, the transgene was detected in 100% (46/46) of embryos analyzed. At d 7, detection frequency was 84% (51/62) in viable embryos, at d 14 49% (18/37), and at d 21 38% (3/8). DNA detection frequency in microinjected bovine embryos by PCR analysis does not give a reliable indication of live transgenic birth rates until after 14 d in culture. Experiment 4 examined microinjected bovine embryos for their potential use as donor embryos in nuclear transfer, or cloning. There was no difference in development between embryos cloned from microinjected donor embryos and those from control donor embryos (injected=11 % (37/377); control=9% (7/81); p>0.05). Of the embryos developing from microinjected donors, 32% (12/37) were PCR positive. Microinjected embryos can be successfully used in a nuclear transfer program to produce more viable embryos, and the resulting embryos may be more reliably screened by PCR. The efficiency of producing viable bovine embryos positive for the injected gene may be increased by performing microinjection early in pronuclear formation, and entering the resulting embryos into a nuclear transfer program. / Ph. D.

The first experiment was conducted to determine the optimum in vitro culture system for one-cell bovine embryos. Subsequent experiments compared bisection and biopsy for acquisition of cellular material from bovine morulae for DNA amplification by the polymerase chain reaction technique (PCR), and evaluated the use of DNA microinjection, in vitro culture, morula bisection, and PCR for production and selection of transgenic bovine preimplantation embryos. In vivo fertilized one-cell bovine embryos were cultured in TCM-199 (n=46), co-cultured with bovine oviductal epithelial cells (OEC; n=38), or in explanted immature mouse oviducts (n=54). Of the embryos that cleaved once, 52.6, and 30.4 and 0.0% developed to morulae/blastocysts after culture in OEC, TCM-199, and explanted mouse oviducts. Mean cell number for embryos cultured in OEC (24.5) was higher than for embryos cultured in TCM-199 (12.8) or in explanted mouse oviducts (5.9; P<.05). Bovine morulae were subjected to bisection (n=44; 20 to 30 cells) or biopsy (n=50; 8 to 12 cells) to assess embryo development in vitro and compare the efficiency of PCR amplification of an endogenous 18S rRNA. Mean development scores (l1=degenerate, 2=morula, 3=blastocyst) and mean cell number after microsurgery and 48 h of culture did not differ between treatments (P>.05; 2.4 ± .1 and 41.8 ± 2.5 versus 2.3 ± .1 and 48.8 ± 2.9, respectively). Frequency of the 18S rRNA amplifications was similar (P>.05) for demi-morulae (78%; 32/41) and biopsies (81%; 39/48). In the third experiment, in vivo fertilized one- (n=155), two- (n=57) and four-cell (n=62) bovine embryos were collected for pronuclear and nuclear DNA microinjection. Approximately 70% of the embryos were injected with DNA and 30% served as controls. Injection did not affect (P>.05) mean development scores after 144 h of cultured in TCM-199 with OEC. Sixty-five (34%) of the DNA injected embryos developed to morulae and were bisected. Injected DNA was amplified by PCR in 29% (19/65) of the demi-morulae. Frequency of DNA detection was more frequent (P<.01) in morulae injected at the 1-cell stage (50%: 16/32) than at the 2-cell (10%; 1/10) or 4-cell (9%: 2/23) stage. Production and selection of transgenic bovine morulae was most successful when one-cell bovine embryos were microinjected. / Ph. D.