Characterizing the relationship between gonadotropin releasing hormone (GnRH), kisspeptin, and RFamide related peptide 3 (RFRP-3) neurons in the equine hypothalamus across the estrous cycle and in the anovulatory seasons.

To understand better the role that kisspeptin plays in regulating seasonal and estrous cycle changes in the mare, this study investigated the number, location and interactions between GnRH, kisspeptin and RFRP-3 neurons in the equine hypothalamus. Hypothalami were collected from mares during the non-breeding season, vernal transition and various stages of the breeding season. Fluorescent immunohistochemistry was used to label the neuropeptides of interest. GnRH cells were observed primarily in the arcuate nucleus (ARC), while very few labeled cells were identified in the pre-optic area (POA). Kisspeptin cells were identified primarily in the ARC, with a small number of cells observed dorsal to the ARC, surrounding the third ventricle (3V). The mean number of kisspeptin cells varied between animals and typically showed no pattern associated with season or stage of estrous cycle, but a seasonal difference was identified in the ARC population. Small numbers of RFRP-3 cells were observed in the ARC, ventromedial hypothalamus (VMH) and dorsomedial hypothalamus (DMH). The mean number of RFRP-3 cells appeared higher in pre-ovulatory animals compared to all other stages. The percentage of GnRH cell bodies with kisspeptin appositions did not change with season or stage of estrous cycle. The percentage of kisspeptin cells receiving inputs from RFRP-3 fibers did not vary with season or stage of estrous cycle. These interactions suggest the possibility of the presence of an ultra-short loop feedback system between these three peptides. The changes in RFRP-3 neurons suggest the possibility of a role in the regulation of reproduction in the horse, but it is unlikely to be as a gonadotropin inhibitory factor.

Design and development of a biological implant for long term intravaginal retention in cattle.

OBJECTIVE: Design, develop and trial an intravaginal device able to remain within the bovine vagina for up to 6 months, accommodating various technologies, while not adversely affecting the cow, or pregnancy. METHODS: Device scoping required minimal interference with vaginal drainage, no constant pressure on any single point of the vaginal mucosa and minimal footprint on the vaginal floor. A polycarbonate prototype was developed consisting of a central cargo space to house sensors and battery, with a separate retention module. Mathematical modelling informed the required length and flexibility of the arms on the retention module to reduce pressure necrosis and inflammation within the vagina. The prototype was initially trialled, with a loaded cargo pod, for 4 weeks in five non-pregnant cows. Cows were monitored for signs of systemic infection and inflammation by collecting blood samples for haematology and acute phase inflammatory proteins. Cows were also assessed for general demeanour, signs of discomfort and positioning of the device. Later, the device was inserted into 20 early pregnant cows, with 20 contemporary, untreated controls. Cows were monitored, as in the initial trial, every 4 weeks for the next 5 to 6 months until calving. RESULTS: In Trial 1, there was 100% retention with no adverse effects. In Trial 2, device retention was 75% until calving with no adverse effects on cow health or pregnancy. CONCLUSIONS: We describe the longest successful deployment of an intravaginal device in pregnant and non-pregnant cattle, with long-term retention, no adverse effects and 28 cm3 of cargo space.

The localization of kisspeptin and kisspeptin receptor in the canine ovary during different stages of the reproductive cycle.

Kisspeptin is a neuropeptide involved in the hypothalamic regulation of reproduction in many species. Recent studies have revealed kisspeptin within the ovaries of rats, Siberian hamsters and humans, indicating a local role in reproduction. However, the role of kisspeptin in the ovary is poorly understood in the bitch. This study investigated the presence and location of kisspeptin protein (KISS1) and kisspeptin receptors (KISS1R) in the canine ovary during different stages of the reproductive cycle (pre-pubertal, anoestrus and cycling) by means of immunohistochemical staining. Ovaries from 24 bitches presented at local veterinary clinics for routine ovariohysterectomy were collected and grouped based on reproductive stage (pre-pubertal, anoestrus and cycling (proestrus, oestrus and dioestrus)). The presence or absence of immunoreactive KISS1 and KISS1R was recorded without any quantification of the levels of expression within cells. Immunoreactive KISS1 was found in the oocytes during all stages of the oestrous cycle, in the granulosa cells during all stages except anoestrus and in the corpus luteum (CL) during dioestrus. KISS1 was absent in the ovaries of pre-pubescent bitches. Immunoreactive KISS1R were consistently found in the oocytes, primordial follicles, the granulosa cells and CL in cycling bitches. The finding of KISS1R in the granulosa cells is suggestive that kisspeptin and progesterone may be linked as this pattern of staining is seen in animals that show preovulatory luteinisation of follicles during oestrus, KISS1R were also observed in the ovaries of pre-pubescent and anoestrous bitches, suggesting a possible role of kisspeptin in oocyte proliferation, development and maturation of granulosa cells, and progesterone production. This study provides a starting point for the establishment of a canine model for kisspeptin regulation within the ovary.

Increased dietary protein in the second trimester of gestation increases live weight gain and carcass composition in weaner calves to 6 months of age.

Genetically similar nulliparous Polled Hereford heifers from a closed pedigree herd were used to evaluate the effects of dietary protein during the first and second trimester of gestation upon foetal, placental and postnatal growth. Heifers were randomly allocated into two groups at 35 days after artificial insemination (35 days post conception (dpc)) to a single bull and fed high (15.7% CP) or low (5.9% CP) protein in the first trimester (T1). At 90 dpc, half of each nutritional treatment group changed to a high- or low-protein diet for the second trimester until 180 dpc (T2). High protein intake in the second trimester increased birth weight in females (P=0.05), but there was no effect of treatment upon birth weight when taken over both sexes. Biparietal diameter was significantly increased by high protein in the second trimester with the effect being greater in the female (P=0.02), but also significant overall (P=0.05). Placental weight was positively correlated with birth weight, fibroblast volume and relative blood vessel volume (P<0.05). Placental fibroblast density was increased and trophoblast volume decreased in the high-protein first trimester treatment group (P<0.05). There was a trend for placental weight to be increased by high protein in the second trimester (P=0.06). Calves from heifers fed the high-protein treatment in the second trimester weighed significantly more on all occasions preweaning (at 1 month (P=0.0004), 2 months (P=0.006), 3 months (P=0.002), 4 months (P=0.01), 5 months (P=0.03), 6 months (P=0.001)), and grew at a faster rate over the 6-month period. By 6 months of age, the calves from heifers fed high nutrition in the second trimester weighed 33 kg heavier than those fed the low diet in the second trimester. These results suggest that dietary protein in early pregnancy alters the development of the bovine placenta and calf growth to weaning.

Reproductive Performance of Arabian and Thoroughbred Mares under Subtropical Conditions of Pakistan.

Breeding records of 57 Arabian and 66 Thoroughbred mares were analysed to assess their reproductive performance under the subtropical conditions of Pakistan. The Arabian mares showed significantly higher conception rates (p<0.05) in second mated oestrus and foal heat mated oestrus compared to Thoroughbred mares. However, conception rates for first lifetime mated oestrus were similar in both breeds of mares. Age at first mating (1,301±40 vs 1,500±32 days) was significantly (p<0.05) less in Arabian compared to Thoroughbred mares. Both breeds of mares showed significantly (p<0.05) higher frequencies of oestrous cycles and conception rates during the winter (October to March) compared to summer (June to August) months. Age of mares affected the conception rates, as mares at ages 3 to 7 and 8 to 12 years of ages had significantly higher conception rates (p<0.05) than those ≥18 years old in both breeds. This study demonstrates that i) reproductive performance in Arabians is better than Thoroughbred mares under the subtropical conditions of Pakistan, ii) mares remain cyclic throughout the year and iii) conception rates were higher in mares bred during winter compared to summer months.

Effects of administration of gonadotropin-releasing hormone at artificial insemination on conception rates in dairy cows.

A controlled trial investigating the effect on conception of administration of 250 µg of gonadotropin-releasing hormone (GnRH) at artificial insemination (AI) in dairy cows in seasonal or split calving herds was conducted. Time of detection of estrus, body condition, extent of estrous expression, treatment, breed, age and milk production from the most recent herd test of the current lactation was recorded. Cows were tested for pregnancy with fetal aging between 35 and 135 days after AI. Sixteen herds provided 2344 spring-calved cows and 3007 inseminations. Logistic regression adjusting for clustering at herd level was used to examine the effect of treatment for first (2344) and second (579) inseminations separately. For first AI, treatment significantly improved conception rate in cows with milk protein concentrations of 3.75% or greater and for cows with milk protein concentrations between 3.00% and 3.50% and less than 40 days calved; increased conception rate from 41.2% to 53.4%. Treatment reduced conception rates in cows with milk protein concentrations of 2.75% or less. Treating only cows identified as responding positively to treatment (11% of all study cows) was estimated to increase first service conception rate in herds from 48.1% to 49.4%. There was no significant effect of treatment on conception to second AI, nor any significant interactions. These findings indicate that GnRH at AI should be limited to the sub-group cows most likely to respond. The positive effect of GnRH at AI may be mediated through improved oocyte maturation and/or improved luteal function, rather than by reducing AI-to-ovulation intervals.

Physiological and behavioral responses of sheep to gaseous ammonia.

Ammonia can accumulate in highly stocked sheep accommodation, for example during live export shipments, and could affect sheep health and welfare. Thus, the objective of this experiment was to test the effects of 4 NH(3) concentrations, 4 (control), 12, 21, and 34 mg/m(3), on the physiology and behavior of wether sheep. Sheep were held for 12 d under a micro-climate and stocking density similar to shipboard conditions recorded on voyages from Australia to the Middle East during the northern hemispheric summer. Ammonia increased macrophage activity in transtracheal aspirations, indicating active pulmonary inflammation; however, it had no effect (P > 0.05) on hematological variables. Feed intake decreased (P = 0.002) in proportion to ammonia concentration, and BW gain decreased (P < 0.001) at the 2 greatest concentrations. Exposure to ammonia increased (P = 0.03) the frequency of sneezing, and at the greatest ammonia concentration, sheep were less active, with less locomotion, pawing, and panting. Twenty-eight days after exposure to NH(3), the pulmonary macrophage activity and BW of the sheep returned to that of sheep exposed to only 4 mg/m(3). It was concluded that NH(3) induced a temporary inflammatory response of the respiratory system and reduced BW gain, which together indicated a transitory adverse effect on the welfare of sheep.

The physiological and behavioral responses of steers to gaseous ammonia in simulated long-distance transport by ship.

Ammonia can accumulate in high-density cattle accommodation during live export shipments and potentially threaten the health and welfare of the animals. The effects of 4 NH(3) concentrations, control (<6), 11, 23, and 34 mg/m(3), on the physiology and behavior of steers were recorded. The animals were held for 12 d under a microclimate and stocking density similar to shipboard conditions experienced on voyages from Australia to the Middle East during the northern hemispheric summer. In bronchoalveolar lavage samples, ammonia increased (P < 0.05) macrophage activity in proportion to NH(3) concentration and increased (P < 0.05) neutrophil percentage at 23 and 34 mg/m(3), indicating active pulmonary inflammation. Ammonia also increased (P < 0.05) lacrimation, nasal secretions, and coughing, particularly at 34 mg/m(3), indicating that the NH(3) was irritating the mucous membranes of the eyes, nasal cavity, and respiratory tract. Ammonia had no effect (P > 0.05) on hematological variables or BW. Twenty-eight days after exposure to NH(3), the pulmonary macrophage activity and neutrophil concentrations of the steers had returned to normal. It was concluded that ammonia concentrations of 23 and 34 mg/m(3) induced temporary inflammatory responses, which indicate an adverse effect on the welfare of steers.

Dystocia in 3-year-old beef heifers; relationship to maternal nutrient intake during early- and mid-gestation, pelvic area and hormonal indicators of placental function.

The influence of nutrition during the first and second trimesters of gestation on the occurrence of dystocia was investigated in 3-year-old composite-breed beef heifers. Heifers were allocated according to stratification by weight and genotype to either a high (H/-=76 MJ metabolisable energy (ME) and 1.4 kg crude protein (CP)), or low (L/-=62 MJ ME and 0.4 kg CP daily) nutritional treatment on the day of artificial insemination (day 0) to the same Senepol bull. Half of each nutritional group changed to an opposite nutritional group on day 93 of gestation (-/H=82 MJ ME and 1.4 kg CP; -/L=63 MJ ME and 0.4 kg CP daily), resulting in four treatment groups: HH (n=16); HL (n=19); LH (n=17); LL (n=19). From 180 days until calving all heifers were fed the same diets. Pelvic area measures were taken at heifer selection (-72 days) and at 117 days. Maternal circulating concentrations of estrone sulphate (ES), bovine placental lactogen (bPL), bovine pregnancy associated glycoprotein and progesterone were monitored throughout gestation. Heifers were observed continuously over the calving period and delivery type classified as being either eutocic or dystocic. The occurrence of dystocia was 14.1%. Increased calf birth weight increased the odds of occurrence of dystocia (odds ratio (OR)=1.40; 95% confidence interval (95% CI) 1.12-1.76; P<0.01). High diets in the second trimester were associated with heavier calves at birth (P=0.01). The mean pelvic area of eutocic heifers on -72 d, tended to be greater compared to that of dystocic heifers (P=0.08) such that a 1-cm(2) difference in pelvic area tended to decrease the risk of dystocia (OR=0.97; 95% CI 0.93-1.01; P=0.09). Longer gestation length was associated with an increased risk of dystocia (P=0.03). ES (P=0.04) and bPL (P=0.09) at calving were positively associated with the risk of dystocia. In conclusion, the current study demonstrates (a) that pelvic area measurement at selection in 3-year-old Bos indicusxBos taurus heifers may be useful for identifying heifers at an increased risk of dystocia and (b) increased ES and bPL concentrations at calving are associated with increased risk of dystocia. Pelvic area measurements obtained prior to conception remain valid in their assessment of the relationship between pelvic area and likelihood of dystocia occurring in the event of changing maternal nutrient intake during gestation. This is an important finding given maternal diets high in protein and energy during the second trimester of gestation increased calf birth weight and calf birth weight was associated with an increase in the occurrence of dystocia in heifers calving as 3-year olds.

Heifer nutrition during early- and mid-pregnancy alters fetal growth trajectory and birth weight.

Maternal nutrient intake during gestation can alter fetal growth. Whilst this has been studied extensively in the sheep, less is known about effects in the bovine. Composite-breed beef heifers were allocated to either a high (H/-=76 MJ metabolisable energy (ME) and 1.4 kg crude protein (CP)) or low (L/-=62 MJ ME and 0.4 kg CP daily) nutritional treatment at artificial insemination. Half of each nutritional group changed to an opposite nutritional group at the end of the first trimester (-/H=82 MJ ME and 1.4 kg CP; -/L=62 MJ ME and 0.4 kg CP daily), resulting in 4 treatment groups: HH (n=16); HL (n=19); LH (n=17); LL (n=19). During the third trimester all heifers were fed the same diets. Fetuses were measured at 4-weekly intervals beginning at day 39 of gestation. Calves were also measured at birth for physical body variables. Low maternal nutrient intake was associated with decreased crown-rump length at day 39 (P<0.01) and increased thoracic diameter at day 95 (P<0.01). Umbilical cord diameter was reduced in L/- fetuses in the first trimester (P<0.05) but was greater in -/L fetuses in the second trimester compared to their respective H counterparts (P<0.05). Calf birth weight was decreased in association with -/L maternal diets (P<0.05). In conclusion, fetal development of cattle may be affected by maternal nutrition as early as day 39 of gestation. This may be followed by either compensatory fetal growth, or alternatively, preferential fetal tissue growth that is dependant upon maternal nutrition. Clearly, calf birth weight may be altered by maternal nutrition during mid-gestation.

Use of vaginal electrical resistance to diagnose oestrus, dioestrus and early pregnancy in synchronized tropically adapted beef heifers.

The primary objective of this study was to determine whether a single measurement of intravaginal electrical resistance (VER), using the commercially available Ovatec probe, can discriminate between dioestrus and oestrus in Bos indicus females, which had been treated to synchronize oestrus. Santa Gertrudis heifers (n = 226) received one of three oestrous synchronization treatments: double PGF(2alpha) 10 days apart, 8-day controlled internal drug release (CIDR) treatment or CIDR pre-synchronization + PGF(2alpha) 10 days after CIDR removal. The heifers were inseminated within 12 h following observed oestrus, or, if not observed, at a fixed time approximately 80 h, following the last synchronization treatment. They were palpated per rectum for signs of pregnancy 9 weeks after artificial insemination (AI). Vaginal electrical resistance measurements were taken at the completion of synchronization treatments (presumed dioestrus), immediately prior to AI (oestrus), and then at 3 and 9 weeks post-AI. Mean VER differed between presumed dioestrus and oestrus (113.7 vs 87.4, p < 0.001). The area under the receiver operating characteristics (ROC) curve was 0.925, indicating that VER was highly discriminatory between dioestrus and oestrus. Vaginal electrical resistance at time of AI was negatively associated with odds of conception when all inseminations were included in the analyses [odds ratio (OR) = 0.97; 95% CI 0.95-1.00; p = 0.018], but not when fixed time AIs were excluded (OR = 1.00; 95% CI 0.97-1.03; p = 0.982). Mean VER readings differed between pregnant and non-pregnant animals at both 3 weeks (120.5 vs 96.7, p < 0.001) and 9 weeks (124.0 vs 100.3, p < 0.001) post-AI. However, 3- and 9-week VER measurements were not highly discriminatory between pregnancy and non-pregnancy (area under ROC curve = 0.791 and 0.736, respectively). Mean VER at time of AI for animals diagnosed in oestrus differed between each of the oestrous synchronization treatments (84.7, 73.6 and 78.9, groups 1-3 respectively, p < 0.001). These findings suggest that measurement of VER may improve accuracy of oestrus diagnoses when selecting cattle for AI following oestrous synchronization programmes involving tropically adapted cattle.

Improved prediction of ovulation time may increase pregnancy rates to artificial insemination in lactating dairy cattle.

A prospective observational study was conducted in two Australian dairy herds to assess the potential for improving pregnancy rates (proportions of inseminations that result in pregnancy) to artificial insemination (AI) if the time of ovulation could be predicted with more certainty. Herd 1 calved year-round and inseminations were performed during two periods each day. Herd 2 calved during autumn-winter and inseminations were performed only after the morning milking each day. In both herds, the AI to ovulation interval of enrolled cows was determined by trans-rectal ovarian ultrasonography approximately 0, 12, 24 and 36 h after AI, and pregnancy was assessed by palpation per rectum 35-56 days after AI. Also, in Herd 1 vaginal electrical resistance (VER) measurements were taken at approximately 0, 12, 24 and 36 h after AI, and in Herd 2 cows were fitted with neck mounted activity meters that monitored cow activity count in 2-h periods. There was substantial variation in the intervals from AI to ovulation within and between herds (mean ± SD 21.2 ± 10.7, n = 102; 14.7 ± 10.4, n = 100 in herds 1 and 2, respectively). Pregnancy rates were higher for inseminations close to, but preceding, ovulation. Using combined herd data (n = 202), the highest pregnancy rate (50.8%) was observed for inseminations between 0 and 16 h before ovulation, a period in which only a modest proportion of inseminations (31.2%) occurred. In contrast, pregnancy rate was significantly lower (28.7%; risk ratio 0.6; 95% CI 0.4-1.0; p = 0.039) for inseminations between 16 and 32 h before ovulation, a period where the highest proportion of inseminations (53.2%) occurred. Thus pregnancy rates could potentially be improved if a greater proportion of inseminations were conducted shortly before ovulation. In Herd 1, mean VER during the peri-ovulatory period varied with time from ovulation. Lowest values (mean ± SEM, VER = 64.8 ± 1.2, n = 55) occurred approximately 18 h before ovulation and were significantly lower than measurements approximately 6 h before ovulation (67.4 ± 1.0; n = 73; p = 0.003). Further work is required to determine if VER can be used to identify ovulation time and hence the optimal time to inseminate in individual animals. In Herd 2 a modest proportion of inseminations (26.9%) occurred between 24 and 40 h after the onset of increased cow activity where the highest pregnancy rate (67.9%) was observed, whereas a significantly lower pregnancy rate (42.4%; risk ratio 0.6; 95% CI 0.4-0.9; p = 0.036) was observed for inseminations between 8 and 24 h after the onset of increased cow activity where the highest proportion of inseminations (56.7%) occurred. Thus cow activity monitoring may be useful to identify the optimal time to inseminate cows. Results from this study indicate that improved methods of ovulation prediction may allow better insemination timing relative to ovulation and consequently increased pregnancy rates.

Insulin-like growth factor levels during pregnancy in the cow are affected by protein supplementation in the maternal diet.

To determine if dietary protein supplementation in early pregnancy alters total circulating insulin-like growth factor (IGF) levels, genetically similar heifers were fed diets containing different levels of protein in the first and second trimesters of gestation. The groups were: low/low (L/L), fed a diet containing 7% crude protein (CP) per kg/DM (low protein) in the first and second trimesters; high/high (H/H), fed a diet containing 14% CP per kg/DM (high protein) in the first and second trimesters; low/high (L/H), fed low protein in the first trimester and high in the second trimester and vice versa for the high/low (H/L) group. At day 62 of gestation, there was a significant difference (P<0.01) in IGF I concentrations between the high and low protein groups (149 versus 119 ng/ml, S.E. 5.9). There was a strong effect (P<0.001) of protein levels in the second trimester on IGF I levels on days 119, 153, and 183 of gestation but not at day 257. Mean IGF I levels for high and low nutrition in the second trimester were 157 and 97 (S.E. 6.6) for days 119, 191, and 88 (S.E. 12.6) for days 153 and 160, and 67 (S.E. 7.7) for day 183. At day 257, there was a significant interaction (P<0.01) between treatments with the means being 98(ab), 110(b), 116(b) and 79(a gamma) (means followed by a letter in common do not differ significantly, P<0.05) (S.E. 7.5) for H/H, H/L, L/H, and L/L, respectively. There was a significant (P<0.05) effect of protein supplementation in the first trimester on calf IGF I levels at birth with means being 42 and 25 (S.E. 5.2) for high and low protein supplementation, respectively. There was a significant (P<0.01) effect of protein supplementation in second trimester upon IGF II levels and a significant (P<0.05) negative correlation between calf birth weight and IGF II levels.

Low dietary protein during early pregnancy alters bovine placental development.

To determine if low dietary protein concentration in the first two trimesters of pregnancy alters placental development, genetically similar heifers from closed herd were fed diets containing different levels of protein in the first and second trimesters of gestation. There were four animals per treatment group, the groups being: L/L = fed a diet containing 7% crude protein (CP) (low protein) in the first and second trimesters; H/H = fed a diet containing 14% CP (high protein) in the first and second trimesters; L/H = fed low protein in the first trimester and high in the second trimester and vice versa for the H/L group. Low protein diets in the first trimester increased dry cotyledon weight at term. Trophectoderm' volume density increased in the H/L and L/H group compared to the L/L and H/H groups. Blood vessel volume and volume density in foetal villi decreased in the H/L and L/H groups compared with the H/H and L/L groups. There was no effect of diet treatment on cotyledon number, diameter or wet weight and no effect on the volume density of connective tissue or fibroblasts in the foetal villi. These results show that a low dietary protein concentration in the first trimester of pregnancy followed by increased protein in the second trimester enhanced placental development. Further, trophectoderm volume was highly correlated with birth weight. Early protein restriction in the pregnant cow may enhance foetal growth in part by stimulating placental growth and function.

Ultrasound-guided cannulation of the caudal vena cava in the bovine for selective sampling of ovarian effluent.

The accuracy of real-time, B-mode ultrasonography was assessed in the visualization and placement of the cannula tip, cranial to the entrance of the ovarian veins as they enter the caudal vena cava of the bovine. A cannula containing a wire guide was introduced into the coccygeal vein via a 14-gauge needle, and was then directed cranially into the caudal vena cava. Once the caudal vena cava was successfully cannulated (7 of 14 cows), ultrasonography allowed for the visualization of the cannula in 7 out of 7 cows. The tip of the cannula was successfully placed cranial to the entrance of the ovarian effuent into the vena cava in 6 of these 7 animals using ultrasound guidance. This was confirmed using progesterone or prostaglandin F(2alpha) radioimmunoassay (RIA). The primary limitation to this technique was the initial catherization of the coccygeal vein which was not achieved in 7 of 14 cows attempted in this study. Successful cannulation was limited to large framed Holstein cows that had at least one calf. Results from this study indicate that real-time, B-mode, ultrasonography is an effective tool for the visualization and accurate placement of the cannula tip within the caudal vena cava. This finding could have implications for research in ovarian hormonal physiology in the cyclic, postpartum or anestrous cow.

Drainage of lymph from the foreleg to the superficial cervical lymph node in sheep.

The superficial cervical (prescapular) node in sheep is large and readily accessible. It lies medial to the omotransverse muscle and 40 to 60 mm cranial to the ventral end of the scapular spine. Lymph vessels reaching this node from the foreleg all enter its ventral half. These lymphatics can be approached surgically opposite the elbow joint where they lie adjacent to the cephalic vein. Carbon particles infused into a single afferent lymphatic were restricted to a small segment of the ventrocranial quadrant of the node. When Evans' Blue dye, or a suspension of carbon particles, was injected subcutaneously just above the hoof they were carried to the node in two to five afferent lymphatics and were found mainly in the ventrocranial quadrant. A knowledge of the anatomy of the superficial cervical node and its afferent lymphatics may be of value in immunological studies, especially where it is desired to introduce antigen into a known part of a node and to leave the remainder for control observations.