The experiments reported in this thesis were conducted to overcome limitations in conventional processes of <i>in vitro-</i>production of embryos and to develop novel procedures for cryopreservation of bovine oocytes and embryos. The experiments, concentrating initially on unfertilized oocytes and subsequently on embryos, used methods relevant to livestock reproductive biotechnology. Key aims were to overcome reliance on media using animal-derived constituents such as serum and albumin. Bovine oocytes matured <i>in vitro</i> in a novel biosafe formulation (MM1mat) excluding serum and albumin reached blastocyst stages (after <i>in vitro</i> fertilization) at rates equivalent to those achieved with conventional protocols (P&gt;0.05). Experiments that investigated development of bovine zygotes in an original series of protein-free culture media (V1 for embryos to Day 4 post-fertilization; V2a and similar formulations for Day 4 onwards) showed that sequential culture in V1/V2a did not compromise development to the blastocyst stage when compared to ‘monoculture’ in an albumin-supplemented medium (P&gt;0.05). Blastocysts produced in V1/V2a survived better after vitrification than counterparts produced in presence of albumin. Vitrification solutions (also often reliant on harmful serum-supplemented media) could be superseded by novel alternatives using V2a as a biohazard-free base medium. It was concluded that bovine blastocysts can be produced and cryopreserved safely in the novel media. A further important aspect of the investigations was that they developed a new biosecure ‘closed system’ for cryopreservation of oocytes and embryos. That system employed the CryoTip –which could be sealed before contact with liquid nitrogen – and the subsequent metabolic and biochemical studies on V1/V2a-produced blastocysts, either fresh or vitrified, indicated pyruvate metabolism, amino acids depletion and peroxide status parameters were equivalent to controls.

The study investigated healthy claw samples from cross-bred heifers aged around 20 months. Histological studies adopted a novel sectioning approach along planes parallel to the wall, identifying dynamics of the keratinisation process, localisation of elastic fibres and anatomical features of cap papillae and horn. Structural molecules were investigated immunohistochemically, including cytokeratins, cytokeratin 14 (CK14) and β-actin. The results showed patterns of suprabasal keratin deposition and loss of signal for CK14 and β-actin markers in differentiating keratinocytes. Localisation of vimentin provided evidence of mesenchymal cells in laminar dermis. Transforming growth factor β1 (TGFβ1) and connective tissue growth factor (CTGF) were investigated immunohistochemically, by <i>in situ</i> hybridisation and RT-PCR. Immunohistochemistry showed signals in the dermis and epidermis, with mRNA presence solely in the dermis and high synthesis rates on comparison with internal control. Mean expression levels were higher in summer than in winter. Collagen I (COL1A2), elastin, estradiol receptor β, cyclin A and B were also investigated by RT-PCR and consistently amplified. Tissue cultures were carried out in order to study TGFβ1, CTGF and COL1A2 expression by RT-PCR in tissue explants incubated in presence of TGFβ1. Results identified a time effect but absence of a concentration effect. Proteomic analysis of fractions extracted from incubated and non-incubated (control) explants showed significant differences for a number of spots, mainly in the keratin region of the electrophoretic gels. In conclusion, the study has provided novel information on molecules of importance in the structure and regulation of epidermal and dermal cells and dermal extracellular matrix in the laminar region of the bovine claw.

Chapter One. A general introduction on feed resources in the coastal lowland Kenya, use of the legume tree forages, and their effect when used as supplements to low quality roughage is presented. This is followed by a review of the literature on the role of microbes in the rumen and the synthesis of microbial protein. The factors that influence the feed intake in ruminants are also reviewed. The role of the leguminous tree forages as supplements to low quality basal diet is presented with more emphasis on the effects of feeding forages from gliricidia and leucaena trees, on intake, digestion and animal productivity. The effects of deleterious compounds with particular reference to tannin is reviewed. Chapter Two. Twenty intact and five fistulated crossbred steers (Ayrshire/Brown Swiss x Sahiwal) were used in Experiment One. The materials and methods used in this experiment had much in common with those of the three following experiments. Therefore procedures similar to all experiments are described in this chapter and only briefly mentioned in the subsequent chapters. The objective of the experiment was to describe the response in production as a result of supplementing napier grass basal diet with incremental levels of gliricidia forage. Napier grass basal diet was offered ad libitum alone or supplemented with 7.5, 15, 22.5 or 30 g DM/kg W0.75/d. Intake and live weight changes were measured for 49 days, and digestibility, and microbial N supply during the last week of the experiment with the twenty steers, in a randomized design. The rumen parameters were measured in the five fistulated steers in a 5 x 5 latin square design. Total Dry matter (DM) intake tended to increase (5.2, 5.1, 5.2, 5.4 and 5.7 kg DM/d, s.e.d 0.21; P 0.05) with supplementation, but this was accompanied by a linear decrease in napier grass intake (5.2, 4.7, 4.5, 4.3 and 4.2 kg DM/d, s.e.d 0.21; P 0.05). The diet digestibility, rumen pH and in sacco DM degradation of napier grass remained unchanged, while the rumen ammonia concentrations (NH3-N) were increased linearly (P 0.05) from 130 to 215 mg/1 for control and highest level of gliricidia forage offered. When gliricidia was offered as proportionally 0.26 of the diet, animal weight gains were increased proportionally by about 0.56. The relationship between the amount of gliricidia offered and the average daily gain was such that, every 10 g DM /kg W0-75 increment of gliricidia forage resulted in 49 g/d of live weight gain. The estimated microbial N supply were lowest in the control group and supplementation tended to increase the yield, but with no significant difference across the treatments. Chapter Three. The objective of Experiment Two was to examine the effect of supplementing napier grass with incremental levels of leucaena forage on voluntary food intake, diet digestibility, rumen fermentation, and live weight gains in steers. The same animals used in Experiment One were used in this experiment. The levels of leucaena offered were 0, 7.5, 15, 22.5 or 30 gDM/kg W0.75/d, the same as those of gliricidia in Experiment One. Increasing the proportion of leucaena forage in the diet of steers offered napier grass had no significant effect on the intake of the napier grass (5.2, 5.3, 5.3, 5.3 and 5.0 kg DM/d, s.e.d. 0.21). The total DM intake increased linearly (5.2, 5.8,6.2,6.6 and 6.7 kg DM/d, s.e.d 0.31; P 0.001). The response was such that for every increment of 10 g DM/kg W0.75 of leucaena, the total intake was increased by 0.52 kg DM/d. Diet digestibility tended to increase, while the rumen pH and in sacco DM degradation characteristics remained unchanged with supplementation. Rumen NH3-N was significantly increased by approximately 11 mg/1 for every 10 g DM/kg W0.75/d of leucaena forage offered. The growth of the animals was improved with supplementation, and the relationship between the amount of leucaena in the diet and the gain was such that for every 10 g DM/kg W0-75/d increment of leucaena forage intake, the gains were increased by 96 g/d. It could be predicted that 1 kg of leucaena offered would result in 171 g/d of gain. At a highest level of leucaena offered ie. proportionally 0.27 of the diet, the weight gains were increased by 0.57 compared with the control group. Chapter Four. The objective of Experiment Three was to examine the effect of incremental levels of gliricidia forage on voluntary food intake, digestion, microbial N supply and live weight gains. Twenty crossbred steers (Ayrshire/Brown Swiss x Sahiwal) and five fistulated steers of the same breed were used for the trial. Intake, diet digestibility, microbial N supply and live- weight changes were measured using the twenty steers and the rumen parameters using the five fistulated steers. Maize stover was offered ad libitum plus 1 kg of maize bran alone, or supplemented with 7.5, 15, 22.5 or 30 g DM/kg W0.75/d of gliricidia forage. Supplementation with gliricidia forage significantly increased the total DM intake (3.0, 3.0, 3.2, 3.5, 3.5 kg DM/d, s.e.d. 0.10; P 0.001) but depressed the intake of maize stover. The response in total DMI was an additional of 0.21 kg DM/d for an increment of 10 g DM/kg W0.75 of the legume. Diet digestibility was not significantly changed, nor was the rumen pH. The degradation parameters was slightly higher in the supplemented group, however, there were no difference in the degradation characteristics of the feeds incubated in steers offered different level of legume forage. Rumen NH3-N and live weight gains were significantly increased with supplementation. The relationship between the proportion of the supplement in the diet and the rumen ammonia or daily gains were such that, for every increment of 10 g DM/kg W0 75 of the legume offered the NH3-N and daily gain were increased by 16 mg/1 and 69 g/d respectively. The microbial N supply tended to be higher in the supplemented groups than the control (30.8, 37.2, 32.9, 34.9, 32.3, s.e.d. 1.06; P 0.05). Chapter Five. Experiment Four compared the type (gliricidia or leucaena) and levels (15 or 30 g DM/kg W0.75) of legume forages given as supplements to maize stover. The effects on intake, diet digestibility, rumen parameters, microbial N supply and live weight gains are presented and discussed. The same steers used in Experiment Three were used in this experiment (Twenty for the growth trial, and the five fistulated animals for the measurements of rumen parameters. The steers were offered maize stover ad libitum plus 1 kg maize bran alone, (2 kg for the fistulated animals), or supplemented with either 15 or 30 g DM/kg W0.75/d of gliricidia or leucaena (Control, G15, G30, L15 or L30). The intake of the basal diet was significantly (P 0.05) increased from 2.3 to 2.5 and 2.3 to 2.7 kg DM/d when of either gliricidia or leucaena respectively were offered as 0.17 of the diet. At higher level of supplementation (to about 0.33 of diet), the maize stover intake tended to decline. The total DM intake were increased (P 0.001) at both levels of both supplements offered (3.2,4.1,4.6,4.3 and 4.6 DM/kg, s.e.d 0.05, being the intakes for control, G15, G30, L15 and L30 respectively). Supplementation at the lower level significantly increased the diet digestibility. The rumen pH remained unchanged, the rumen NH3-N increased (31, 80, 101, 95, 111 mg/1 s.e.d 20.4; P 0.001), and the in sacco DM characteristics of the feeds tended to increase with supplementation. Both the DM and nitrogen (N) in leucaena forage were degraded at a slower rate than in gliricidia. The control group had the lowest (P 0.001) gains, compared with the supplemented groups. The leucaena supplemented group tended to have higher live weight gains than the gliricidia group, but the differences were not significant. Gains were 81, 355, 695, 396, 753 g/d s.e.d 44.5 for control, G15, G30, L15 and L30 groups respectively. With all parameters measured, there were no significant difference between supplementation with gliricidia or leucaena forages, at either level.

The specification of shelter requirements of cattle requires knowledge of the complex interactions of climate, the enclosed animals and their enclosure. The modelling approach used in this research reduces this complexity by identifying the most significant components of the system. Four electrically heated physical thermal models of suckler cows with controlled internal temperatures and coverings of synthetic hair were used over two consecutive winter periods to simulate sensible heat losses from suckler cows kept in an unsheltered site and in three simple shelters. The measured reductions of unsheltered heat losses were (1) 2% (2) 6% and (3) 20% for shelters with respectively (1) just a roof (2) four solid walls, no roof and (3) a roof and four walls with ventilation openings. A mathematical model which predicts hourly and daily mean sensible heat losses from a suckler cow using measurements of air temperature, rainfall, windspeed and net radiation had previously been developed using a large thermal model. Climate modifying factors were derived for each shelter type by comparing hourly and daily mean measured heat losses with those predicted by the mathematical model. These factors were successfully validated using the second winter's data. The accuracies of heat loss predictions for the small thermal models in each shelter were similar to those for the original large thermal model. Examples are given for cattle sheltered by a forest and for calves sheltered by a creep to show how this model for prediction of heat losses in different shelters can be incorporated in a model of the whole system. Meteorological Office climate data was used to predict heat losses from cattle. This included a novel method for prediction of net radiation. The analysis indicated that predictions for a given site could be applied to a large area although this should be tested before any large scale analysis of records is undertaken.

Embryo transfer plays a very important role in the cattle industry and its application requires a consistent supply of viable embryos for use in such programmes. One way of achieving this is the development of reliable superovulation regimens, yielding a large number of high quality embryos. Superovulation with pregnant mare serum gonadotrophin (PMSG) induces a second wave of follicles after ovulation because it is eliminated slowly from the peripheral blood, causing high concentrations of oestradiol. This oestradiol may have an adverse effect on fertilization and early embryonic development. Administering PMSG antiserum after ovulation may improve the quality by neutralizing PMSG activity. The object of this study was to examine the role of monoclonal anti- PMSG (Neutra-PMSG; Intervet UK) in a superovulatory regime for cattle based on PMSG with the objective of increasing the number of viable embryos produced. Two experiments were conducted in this study. In experiment 1, cows were superovulated with 2500 iu of PMSG (Folligon; Intervet UK) im on day 101 of their oestrous cycle, whereas in experiment 2, heifers were superovulated with 1000, 2000, 3000 or 4000 iu of PMSG (PMSG1, 2, 3 or 4, respectively) im on day 10 1 of their oestrous cycle. In experiments 1 and 2, animals were given 2 ml of PG (PG3) im 48 h after PMSG injection and oestrus was observed in the same manner as described above. When data were evaluated in respect of the PMSG/APMSG dose level, there were no significant differences (P>0.05) in the numbers of CL and usable number of embryos between APMSG treatment and the appropriate control. Treatment with APMSG in the 3000 iu PMSG dose group reduced (P 0.05) the numbers of LF compared to control (1.3 v 5.5). When data were analysed based on the dose levels of PMSG, the total number of ova/embryos and the number of usable embryos were higher in heifers which received 2000 iu of PMSG compared to those which received 1000, 3000 or 4000 iu (7.1 v 2.1, 6.6 or 5.6 and 6.3 v 2.1, 4.8 or 3.2, respectively) . In conclusion the results indicate that PMSG dose 2000 iu is the favoured dose for superovulating heifers. The administration of Neutra-PMSG 36, 48, 60, 72, 84 or 96 h after PG3 injection, despite reducing LF numbers and preventing the rise in 0E2 after ovulation, had no significant effect on the number of usable embryos recovered.

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