Babesia bovis: successful vaccination against homologous challenge in splenectomised calves using a fraction of haemagglutinating antigen

A soluble extract from lysed bovine erythrocytes infected with Babesia bovis, was separated by preparative agarose electrophoresis. A fraction with a mobility of plasma β globulins was shown to contain babesial antigen confined mainly to the infected erythrocyte and was used to vaccinate a group of four calves. Following challenge with homologous B. bovis all four calves vaccinated with the antigen survived the infection whereas all the calves in a control group of four died from infection.     

Trypanosoma evansi: Therapeutic efficacy of diminazine aceturate in crossbred calves, Bos taurus and B. indicus

The therapeutic efficacy of diminazine aceturate (Berenil) against Trypanosoma evansi infection (surra) in calves was determined. Seven crossbred calves, 8 to 12 months old, were inoculated subcutaneously with 5 × 10⁷ parasites of a virulent strain of T. evansi. All of the calves developed clinical infection and exhibited symptoms associated with chronic surra. Forty-one to forty-four days after inoculation, five of the calves were treated with a single dose of diminazine aceturate at 10 mg/kg body weight intramuscularly. The two remaining calves were left as infected but untreated controls. The treated calves were found free of the infection on repeated blood smear examinations and diagnostic challenge tests in mice 12 hr to 16 days later. One of the five calves, which was treated at an advanced stage of the disease, died on the fifth day after treatment. Splenectomy of the treated calves, 16 to 19 days after treatment, did not result in relapse of parasitemia, indicating that diminazine aceturate at 10 mg/kg had a rapid trypanocidal effect in surra of calves. Both of the controls died of the disease on the 45th and 47th day.     

Babesia argentina: Disseminated intravascular coagulation in acute infections in splenectomized calves

The pathology of severe Babesia argentina infections in splenectomized calves was studied. The calves were infected by intravenous inoculation of 10⁹–10¹⁰B. argentina and given 0.1 mg/kg betamethasone to enhance the parasitemia. Hematological changes observed during detailed studies of the course of infection in eight calves, three of which subsequently died, included thrombocytopenia, leucopenia, and reduced fibrinogen levels. The prothrombin time and partial thromboplastin time were prolonged in all three calves tested, and pathological levels of fibrinogen degradation products were detected in both of two calves tested. Massive pulmonary edema was a constant finding at autopsy of 24 fatal cases. Histopathological examination revealed widespread fibrin thrombi in capillaries and larger vessels of lung, in capillaries of renal glomeruli, and in hepatic sinusoids. The findings established the occurrence of disseminated intravascular coagulation in the acute infections studied.     

Oral Administration of Faecalibacterium prausnitzii Decreased the Incidence of Severe Diarrhea and Related Mortality Rate and Increased Weight Gain in Preweaned Dairy Heifers

Probiotics are a promising alternative to improve food animal productivity and health. However, scientific evidence that specific microbes can be used to benefit animal health and performance is limited. The objective of this study was to evaluate the effects of administering a live culture of Faecalibacterium prausnitzii to newborn dairy calves on subsequent growth, health, and fecal microbiome. Initially, a safety trial was conducted using 30 newborn bull calves to assess potential adverse effects of the oral and rectal administration of F. prausnitzii to neonatal calves. No adverse reactions, such as increased body temperature or heart and respiratory rates, were observed after the administration of the treatments. All calves survived the experimental period, and there was no difference in fecal consistency score, attitude, appetite or dehydration between the treatment groups. The rectal route was not an efficient practice while the oral route ensures that the full dose is administered to the treated calves. Subsequently, a randomized field trial was completed in a commercial farm with preweaned calves. A total of 554 Holstein heifers were assigned to one of two treatment groups: treated calves (FPTRT) and non-treated calves (control). Treated calves received two oral doses of F. prausnitzii, one at treatment assignment (1^st week) and another one week later. The FPTRT group presented significantly lower incidence of severe diarrhea (3.1%) compared with the control group (6.8%). Treated calves also had lower mortality rate associated with severe diarrhea (1.5%) compared to control calves (4.4%). Furthermore, FPTRT calves gained significantly more weight, 4.4 kg over the preweaning period, than controls calves. The relative abundance of F. prausnitzii in the fecal microbiota was significantly higher in the 3^rd and 5^th weeks of life of FPTRT calves than of the control calves, as revealed by sequencing of the 16S rRNA gene. Our findings showed that oral administration of F. prausnitzii improves gastrointestinal health and growth of preweaned calves, supporting its use as a potential probiotic.     

Outbreak of cryptosporidiosis due to Cryptosporidium parvum subtype IIdA19G1 in neonatal calves on a dairy farm in China

Neonatal diarrhea is one of the most important syndromes in dairy cattle. Among enteropathogens, Cryptosporidium spp. are primary causes of diarrhea, but outbreaks due to cryptosporidiosis are rarely reported in cattle. From January to April in 2016, severe diarrhea was observed in over 400 neonatal dairy calves on a large dairy farm in Jiangsu Province of East China. Approximately 360 calves died due to watery diarrhea despite antibiotic therapy. In this study, 18 fecal specimens were collected from seriously ill calves on this farm during the diarrhea outbreak, and analysed for common enteropathogens by enzymatic immunoassay (EIA). In a post-outbreak investigation, 418 and 1372 specimens collected from animals of various age groups were further analysed for rotavirus and Cryptosporidium spp. by EIA and PCR, respectively, to assess their roles in the occurrence of diarrhea on the farm. Cryptosporidium spp. were genotyped using established techniques. Initial EIA tests showed that 15/18 seriously ill calves during the outbreak were positive for Cryptosporidium parvum, while 8/18 were positive for rotavirus. The overall infection rate of Cryptosporidium in pre-weaned calves on the farm was 22.7%, with odds of the Cryptosporidium infection during the outbreak 4.4–23.5 times higher than after the outbreak. Four Cryptosporidium spp. were identified after the outbreak including C. parvum (n = 79), Cryptosporidium ryanae (n = 48), Cryptosporidium bovis (n = 31), and Cryptosporidium andersoni (n = 3), with co-infections of multiple species being detected in 34 animals. Infection with C. parvum (73/79) was found in the majority of calves aged ≤3 weeks, consistent with the age of ill calves during the outbreak. All C. parvum isolates were identified as subtype IIdA19G1. In the post-outbreak investigation, C. parvum infection was associated with the occurrence of watery diarrhea in pre-weaned calves, C. ryanae infection was associated with moderate diarrhea in both pre- and post-weaned calves, while no association was identified between rotavirus infection and the occurrence of diarrhea. Results of logistic regression analysis further suggested that C. bovis infection might also be a risk factor for moderate diarrhea in calves. Thus, we believe this is the first report of a major outbreak of severe diarrhea caused by C. parvum IIdA19G1 in dairy calves. More attention should be directed toward preventing the dissemination of this virulent subtype in China.     

Expression and methylation status of imprinted genes in placentas of deceased and live cloned transgenic calves

Placental deficiencies are linked with developmental abnormalities in cattle produced by somatic cell nuclear transfer (SCNT). To investigate whether the aberrant expression of imprinted genes in placenta was responsible for fetal overgrowth and placental hypertrophy, quantitative expression analysis of six imprinted genes (H19, XIST, IGF2R, SNRPN, PEG3, and IGF2) was conducted in placentas of: 1) deceased (died during perinatal period) transgenic calves (D group, n = 4); 2) live transgenic calves (L group, n = 15); and 3) conventionally produced (control) female calves (N group, n = 4). In this study, XIST, PEG3 and IGF2 were significantly over-expressed in the D group, whereas expression of H19 and IGF2R was significantly reduced in the D group compared to controls. The DNA methylation patterns in the differentially methylated region (DMR) from H19, XIST, and IGF2R were compared using Bisulfite Sequencing PCR (BSP) and Combined Bisulfite Restriction Analysis (COBRA). In the D group, H19 DMR was significantly hypermethylated, but XIST DMR and IGF2R ICR were significantly hypomethylated compared to controls. In contrast, there were no noticeable differences in the expression and DNA methylation status of imprinted genes (except DNA methylation level of XIST DMR) in the L group compared to controls. In conclusion, altered DNA methylation levels in the DMRs of imprinted genes in placentas of deceased transgenic calves, presumably due to aberrant epigenetic nuclear reprogramming during SCNT, may have been associated with abnormal expression of these genes; perhaps this caused developmental insufficiencies and ultimately death in cloned transgenic calves.     

Avoidance of roads and selection for recent cutovers by threatened caribou: fitness-rewarding or maladaptive behaviour?

The impact of anthropogenic disturbance on the fitness of prey should depend on the relative effect of human activities on different trophic levels. This verification remains rare, however, especially for large animals. We investigated the functional link between habitat selection of female caribou (Rangifer tarandus) and the survival of their calves, a fitness correlate. This top-down controlled population of the threatened forest-dwelling caribou inhabits a managed forest occupied by wolves (Canis lupus) and black bears (Ursus americanus). Sixty-one per cent of calves died from bear predation within two months following their birth. Variation in habitat selection tactics among mothers resulted in different mortality risks for their calves. When calves occupied areas with few deciduous trees, they were more likely to die from predation if the local road density was high. Although caribou are typically associated with pristine forests, females selected recent cutovers without negative impact on calf survival. This selection became detrimental, however, as regeneration took place in harvested stands owing to increased bear predation. We demonstrate that human disturbance has asymmetrical consequences on the trophic levels of a food web involving multiple large mammals, which resulted in habitat selection tactics with a greater short-term fitness payoff and, therefore, with higher evolutionary opportunity.     

Effects of isobutyrate supplementation in pre- and post-weaned dairy calves diet on growth performance,rumen development,blood metabolites and hormone secretion

Isobutyrate supplements could improve rumen development by increasing ruminal fermentation products, especially butyrate, and then promote the growth performance of calves. The objective of this study was to evaluate the effects of isobutyrate supplementation on growth performance, rumen development, blood metabolites and hormone secretion in pre- and post-weaned dairy calves. In total, 56 Chinese Holstein male calves with 30 days of age and 72.9±1.43 kg of BW, blocked by days of age and BW, were assigned to four groups in a randomized block design. The treatments were as follows: control, low-isobutyrate, moderate-isobutyrate and high-isobutyrate with 0, 0.03, 0.06 and 0.09 g isobutyrate/kg BW per calf per day, respectively. Supplemental isobutyrate was hand-mixed into milk of pre-weaned calves and the concentrate portion of post-weaned calves. The study consisted of 10 days of an adaptation period and a 50-day sampling period. Calves were weaned at 60 days of age. Seven calves were chosen from each treatment at random and slaughtered at 45 and 90 days of age. BW, dry matter (DM) intake and stomach weight were measured, samples of ruminal tissues and blood were determined. For pre- and post-weaned calves, DM intake and average daily gain increased linearly (P<0.05), but feed conversion ratio decreased linearly (P<0.05) with increasing isobutyrate supplementation. Total stomach weight and the ratio of rumen weight to total stomach weight tended to increase (P=0.073) for pre-weaned calves and increased linearly (P=0.021) for post-weaned calves, whereas the ratio of abomasum weight to total stomach weight was not affected for pre-weaned calves and decreased linearly (P<0.05) for post-weaned calves with increasing isobutyrate supplementation. Both length and width of rumen papillae tended to increase linearly for pre-weaned calves, but increased linearly (P<0.05) for post-weaned calves with increasing isobutyrate supplementation. The relative expression of messenger RNA for growth hormone (GH) receptor and 3-hydroxy-3-methylglutaryl-CoA synthase 1 in rumen mucosa increased linearly (P<0.05) for pre- and post-weaned calves with increasing isobutyrate supplementation. Blood concentrations of glucose, acetoacetate, β-hydroxybutyrate, GH and IGF-1 increased linearly (P<0.05) for pre- and post-weaned calves, whereas blood concentration of insulin decreased linearly with increasing isobutyrate supplementation. The present results indicated that isobutyrate promoted growth of calves by improving rumen development and its ketogenesis in a dose-dependent manner.     

Impact of transgenic oilseed rape expressing oryzacystatin-1 (OC-1) and of insecticidal proteins on longevity and digestive enzymes of the solitary bee Osmia bicornis

The risk that insect-resistant transgenic plants may pose for solitary bees was assessed by determining longevity of adult Osmia bicornis (O. rufa) chronically exposed to transgenic oilseed rape expressing oryzacystatin-1 (OC-1) or to the purified insecticidal proteins recombinant rOC-1, Kunitz soybean trypsin inhibitor (SBTI), Galanthus nivalis agglutinin (GNA), or Bacillus thuringiensis toxin Cry1Ab dissolved in sugar solution (at 0.01 and 0.1%, w:v, Cry1Ab only at 0.01%). Compared to control bees, longevity was significantly reduced by SBTI and GNA at both concentrations and by rOC-1 at 0.1%, but not by Cry1Ab or rOC-1 at 0.01%. Longevity on the OC-1 oilseed rape was not significantly different from the control plants. The effects of SBTI and rOC-1 on longevity were investigated through characterization of the digestive proteinases of O. bicornis and analysis of the response in proteinase profiles to ingestion of these proteinase inhibitors. A relatively complex profile of at least four types of soluble proteolytic enzymes was identified. Serine proteinases were found to be predominant, with metallo and especially cysteine proteinases making a smaller albeit significant contribution. The compensatory response to in vivo enzyme inhibition was similar for SBTI and rOC-1 although less pronounced for rOC-1. It consisted of a non-specific overproduction of native proteinases, both sensitive and insensitive, and the induction of a novel aspartic proteinase.     

Effect of rumen-protected methionine supplementation to beef cows during the periconception period on performance of cows,calves, and subsequent offspring

Maternal nutrition affects the development of the fetus and postnatal performance of the calf. Methionine may play a critical role in developmental programming and is likely deficient in beef cows fed low-quality forage. The objective of this study was to determine the effect of metabolizable methionine supply to lactating beef cows during the periconception period on performance of cows, calves, and subsequent offspring. This project involved two consecutive production cycles commencing at calving in which dietary treatments were fed to cows during the periconception period along with measurements on cows and initial calves in Production Cycle 1, and measurements on subsequent calves in Production Cycle 2. Brangus-Angus crossbred lactating beef cows (N = 108; age = 6.4 (2.8) year) were stratified by previous calving date and assigned to one of three supplements: (1) control, molasses plus urea at 2.72 kg/day as fed, (2) fishmeal, 2.27 kg/day molasses plus urea plus 0.33 kg/day as fed of fishmeal, and (3) methionine, 2.72 kg/day of molasses plus urea plus 9.5 g/day of 2-hydroxy-4-(methylthio)-butanoic acid. Cows were fed supplements and low-quality limpograss (Hemarthria altissima) hay while grazing dormant bahiagrass (Paspalum notatum Flüggé) pastures during the 115-day periconception period from December 2014 to April 2015 in Production Cycle 1 only. Body weight change and milk yield of cows were measured during the periconception period in Production Cycle 1. Body weight of calves was measured at birth and weaning in both production cycles. Following weaning in Production Cycle 2, eight subsequent steer calves per treatment were individually housed for a 42-day metabolism experiment. Treatment did not affect (P > 0.10) BW change of cows, but cows fed methionine tended (P = 0.09) to produce more energy-corrected milk than control and fishmeal. Treatment did not affect (P > 0.10) 205-day adjusted weaning weight of calves in either production cycle. During the metabolism experiment, subsequent calves from dams fed fishmeal and methionine gained faster (P < 0.05) and had greater (P < 0.05) gain:feed than control calves. Methionine calves tended (P = 0.06) to have greater apparent total tract NDF and ADF digestibility and lesser (P < 0.05) blood glucose concentration than control and fishmeal calves. These data indicate that maternal methionine supply during the periconception period plays an important role in programming future performance of the offspring.     

Efficacy of Monensin Against Bovine Coccidiosis in Young Holstein-Friesian Calves*

Monensin, an anticoccidial antibiotic, was incorporated into pelleted feed and given to 10-week-old Holstein-Friesian calves at 0.25 mg/kg, 1.0 mg/kg, or 2.0 mg/kg of body weight. Inoculated calves were inoculated by drenching with 500,000 sporulated oocysts of Eimeria bovis. Other calves served as inoculated or uninoculated controls. Observations were recorded on oocyst discharge in the feces, clinical signs, weight gain, food consumption, hemoglobin, packed cell volume, total serum protein, sodium and potassium content of the serum, and differential white cell counts. Calves in the inoculated control group developed severe infections, discharged large numbers of oocysts, developed clinical signs and 1 of 5 died. Uninoculated, untreated control calves were essentially free of coccidia. A few calves in the groups which received monensin developed light infections but none of them had clinical signs of coccidiosis. Calves which received the highest and the lowest dosages of monensin gained weight less rapidly than did the uninoculated controls or the animals which received monensin at 1.0 mg/kg of body weight. Inoculated control calves with severe infections had reduced food intake and a significant reduction in weight which was not regained during the experimental period. The only other significant change in any of the parameters measured was a reduction in the total serum protein of inoculated, nonmedicated control calves. The level returned to normal 5 weeks after clinical signs first appeared.     

Effect of Milk Fermented with Lactic Acid Bacteria on Diarrheal Incidence,Growth Performance and Microbiological and Blood Profiles of Newborn Dairy Calves

The effect of the administration of milk fermented with lactic acid bacteria to calves was evaluated. The strains included were: Lactobacillus murinus CRL1695, Lact. mucosae CRL1696, Lact. johnsonii CRL1693, and Lact. salivarius CRL1702, which were selected for their beneficial and functional properties and isolated from healthy calves in the northwestern region of Argentina. The trial was conducted on a dairy farm located in Tucumán (Holando-Argentino calves). A randomized controlled trial was performed in which 56 new-born animals were divided into two groups: the treated group (T) received the fermented milk for 60 days and the control group (C) only milk. The animals were fed a solid diet ad libitum. The treated group was given a daily dose of 1 × 10⁹CFU of the probiotic fermented milk while the control group was fed milk. Body weight and biometrical parameters were recorded between 15 and 60 days of age, and average daily gain was calculated with three samplings per animal throughout the trial. Rectal swabs and fecal and blood samples were also collected. Results showed the efficacy of the probiotic: lower morbidity and mortality of calves (morbidity was 69.20% in animals without the probiotic, and 46.15% in probiotic-treated animals, with P = 0.09; mortality in C was 34.61 and 7.69% in animals fed with ferment milk; P = 0.02).The calves fed with probiotic evidenced an improvement in nutritional parameters, body condition and weight gain (health index P = 0.01; average daily gain P = 0.03).Viable bacterial numbers showed no differences between the two experimental groups. Hematological parameters and serum proteins were not modified by the treatment. The results suggest that the fermented milk containing lactic acid bacteria can be a viable veterinary product for young calves due to its beneficial effects on health and growth.     

Influence of spray-dried rumen fluid supplementation on performance,blood metabolites and cytokines in suckling Holstein calves

Rumen fluid from slaughtered animals is one of the wastes of slaughterhouses released to the environment that, due to its high nitrogen and phosphorus contents, can lead to soil and groundwater pollution. Meanwhile, it contains ruminal microbes and some bioactive compounds such as enzymes, minerals, vitamins and organic acids. This study was designed to examine the potential of rumen fluid as a feed additive. Therefore, the effects of spray-dried rumen fluid (SDR) with 1% maltodextrin on the performance, blood metabolites and some cytokines of sucking dairy calves during the pre-weaning phase were investigated. Forty male Holstein calves, with a mean weight of 39.4 ± 3.7 kg and 7 ± 1 days old, were randomly assigned to four groups (n = 10 calves per group) in a completely randomized design. Experimental treatments were: control diet with no additive (CON); control diet with 0.5 g/day of SDR (SDR0.5); control diet with 1 g/day of SDR (SDR1); and control diet with 1.5 g/day of SDR (SDR1.5). Daily feed intake and average daily gain of calves were not affected by feeding SDR as a feed additive. Cholesterol concentration was significantly affected by the 20th and 40th days of the experiment and decreased linearly by increasing SDR feeding level. Levels of liver enzymes, including aspartate aminotransferase and alanine aminotransferase, in the blood decreased by feeding SDR at day 40 of the experiment. Serum concentration of interleukin-6 at day 20 was not affected by dried rumen fluid feeding, whereas at day 40, a significant effect was observed among experimental treatments. The lowest value was recorded for SDR1.5 v. control calves. At day 20, the serum concentration of interferon-γ was influenced by supplementing SDR, and the highest value was recorded for SDR1.5 calves. The inclusion of SDR with 1% maltodextrin in suckling dairy calves had beneficial effects on the stimulation of calves’ immune system.     

Effect of a topical anaesthetic formulation on the cortisol response to surgical castration of unweaned beef calves

Impracticality and cost of existing pain management strategies during surgical castration of beef cattle have limited their widespread implementation on-farm. A farmer-applied topical anaesthetic formulation, originally developed and used commercially to mitigate the pain of mulesing in lambs, was investigated for its potential use for managing pain in surgically castrated calves. This formulation contained lidocaine, bupivacaine, adrenalin and cetrimide. In this study, 24 Angus bull calves were randomly allocated to (1) surgical castration (C, n=8), (2) surgical castration with the post-operative application of topical anaesthetic (CTA, n=8) and (3) sham castration/control (CON, n=8). The experiment was conducted over 2 days, with treatment groups evenly represented across each day. Calves were habituated to handling before the experiment and blood samples were collected for plasma cortisol measurement at defined time periods before, at and post treatment, (at −0.5, 0 h, then +0.5, 1, 1.5, 2, 4 and 6 h). There was a significant effect of time on cortisol concentrations across all treatment groups (P<0.01), with lowest concentrations at −0.5 and 6 h and peak concentration at 0.5 h being significantly higher than the cortisol response at 0 h. The effect of treatment was not significant (P=0.077), however, there was a trend for CON calves to display lower cortisol concentrations than C and CTA calves and CTA calves to display lower cortisol concentrations than C calves. The mean area under the curve (AUC) of CON calves was significantly lower than those of C and CTA calves (P=0.04), however, there was no significant difference between the AUCs of CTA and C calves. Immediate application of topical anaesthetic after surgical castration did not significantly reduce plasma cortisol concentrations. However, the trend for CTA calves to display lower cortisol concentrations than C calves warrants further investigation into the use of TA for pain relief of surgically castrated beef calves.     

Extracellular proteinases from the phytopathogenic fungus Fusarium culmorum

The growth of Fusarium culmorum fungus on a medium containing thermostable proteins from potato tubers was accompanied by the production of proteinases, exhibiting activity over a broad pH range (from 6.0–10.0). When studied by SDS-PAGE in the presence of β-mercaptoethanol, extracellular proteinases were represented by at least five species with a molecular weight of 30–60 kDa. Inhibitor analysis and studies of enzyme activities with synthetic substrates demonstrated that the culture liquid of Fusarium culmorum contained serine proteinases of various classes. The amount of subtilisin-like proteinases was the highest. A near-complete inhibition of the enzymes was caused by proteinaceous proteinase inhibitors from potato tubers. These data suggest that proteinases of the phytopathogen Fusarium culmorum serve as a metabolic target for natural inhibitors of potato proteinases.     

Successful low dose insemination of flow cytometrically sorted Sika (Cervus nippon) sperm in Wapiti (Cervus elaphus)

The purpose of this study was to determine a practical method in Wapiti (Cervus elaphus) of using predetermined sexed Sika (Cervus nippon) semen. Semen was collected by electro-ejaculation from one stag of proven fertility and transported to the laboratory where it was retained as unsorted (control) or was separated into X- and Y-chromosome-bearing sperm using a modified high-speed cell sorter. Wapiti hinds (n = 81) were inseminated into the uterus by rectum manipulation with 1 × 10⁶ (X1 and Y1 group, respectively) or 2 × 10⁶ (X2 and Y2 group, respectively) of sorted frozen-thawed and 1 × 10⁷ non-sorted frozen-thawed (a commercial dose control) Sika motile sperm 60–66 h after removal of intra-vaginal progesterone-impregnated CIDR devices and administration of 700 IU of PMSG at the time of CIDR removal. The percentage of hinds calving after insemination was similar for X1 (38.5%), X2 (41.7%), Y1 (44.4%), Y2 (38.9%) groups (P > 0.05), but higher for control (75%) treatment (P < 0.05). Ultimately 15 out of the 16 Sika and Wapiti-hybrid calves produced by Wapiti hinds inseminated with Y-sorted sperm were male (93.7%) and 10/10 (100%) Sika and Wapiti-hybrid calves from hinds inseminated with X-sorted sperm were female. The sex ratio of the Sika and Wapiti-hybrid calves born to hinds inseminated with sex-sorted sperm deviated significantly (P < 0.05) from 50% and 50.0% in the control group. All Sika and Wapiti-hybrid calves were born between 237 and 250 d of gestation. Male and female calves in the control group had similar birth weights and weaning weights as calves from hinds inseminated with X- or Y-sorted sperm. In conclusion it can be said that normal Sika and Wapiti-hybrid calves of predicted sex can be produced after artificial insemination of Wapiti does with low numbers of sex-sorted cryopreserved Sika sperm.     

Pregnancy rate and birth rate of calves from a large-scale IVF program using reverse-sorted semen in Bos indicus, Bos indicus-taurus, and Bos taurus cattle

Obtaining sexed sperm from previously frozen doses (reverse-sorted semen [RSS]) provides an important advantage because of the possibility of using the semen of bulls with desired genetic attributes that have died or have become infertile but from whom frozen semen is available. We report the efficiency of RSS on the pregnancy rate and birth rate of calves in a large-scale program using ovum pick-up and in vitro embryo production (IVEP) from Bos indicus, Bos indicus-taurus, and Bos taurus cattle. From 645 ovum pick-up procedures (Holstein, Gir, and Nelore), 9438 viable oocytes were recovered. A dose of frozen semen (Holstein, Nelore, Brahman, Gir, and Braford) was thawed, and the sperm were sex-sorted and cooled for use in IVF. Additionally, IVF with sperm from three Holstein bulls with freeze-thawed, sex-sorted (RSS) or sex-sorted, freeze-thawed (control) was tested. A total of 2729 embryos were produced, exhibiting a mean blastocyst rate of 29%. Heifers and cows selected for adequate body condition, estrus, and health received 2404 embryos, and 60 days later, a 41% average pregnancy rate was observed. A total of 966 calves were born, and 910 were of a predetermined sex, with an average of 94% accuracy in determining the sex. Despite the lower blastocyst rate with freeze-thawed, sex-sorted semen compared with sex-sorted semen, (P < 0.05), the pregnancy rate (bull I, 45% vs. 40%; II, 35% vs. 50%; and III, 47% vs. 48% for RSS and control, respectively; P > 0.05) and sex-sorted efficiency (bull I, 93% vs. 98%; II, 96% vs. 94%; and III, 96% vs. 97% for RSS and control, respectively; P > 0.05) were similar for each of the three bulls regardless of the sperm type used in the IVF. The sexing of previously frozen semen, associated with IVEP, produces viable embryos with a pregnancy rate of up to 40%, and calves of the desired sex are born even if the paternal bull has acquired some infertility, died, or is located a long distance from the sexing laboratory. Furthermore, these data show the feasibility of the process even when used in a large-scale IVEP program.     

Extended transition milk feeding for 3 weeks improves growth performance and reduces the susceptibility to diarrhea in newborn female Holstein calves

Dairy calves may benefit from extending the duration of feeding transition milk (TM; the subsequent two to six milkings after parturition) to enhance performance and health during early life. The objective of this study was to assess the effect of replacing pasteurized waste milk (non-saleable milk containing antibiotic and/or drug residues) with pasteurized TM for 3 weeks on the growth performance and health of dairy calves. A total of 84 healthy newborn female Holstein calves were blocked by birth order and assigned randomly to 4 treatment groups with partial replacement of pasteurized waste milk by TM (second milkings after parturition) at 0 (0 l/day TM + 6 l/day milk), 0.5 (0.5 l/day TM + 5.5 l/day milk), 1 (1 l/day TM + 5 l/day milk), or 2 l (2 l/day TM + 4 l/day milk) for a 21-day period. From day 22 onward, all calves were fed individually with 6 l/day pasteurized waste milk. Calves were weaned on day 60 and monitored until day 90 of the study. Liquid feed DM intake (DMI) was increased with increasing levels of TM (P = 0.001). Starter feed DMI and total DMI (liquid feed DMI + starter feed DMI) were not affected by the treatment effect. Calves were fed 2 l/day TM gained more BW compared with those in the control group during the postweaning and overall periods. The average daily gain tended (P = 0.06) to be higher in calves fed 2 l/day TM compared with calves fed 0 (+ 65 g/day), 0.5 (+ 53 g/day), or 1 (+ 76 g/day) l/day TM during the preweaning period. Daily weight gain was also higher in calves fed 2 l/day TM compared with calves in the control group during the postweaning (+ 137 g/day; P = 0.04) and overall (+ 89 g/day; P < 0.01) periods, respectively. Calves fed 2 l/day TM had a higher feed efficiency compared with calves in the control group during all studied periods. The calves fed TM2 had a lower chance of having diarrhea compared with other treatment groups. Duration but not the frequency of diarrhea was lower in calves fed TM2 vs TM0 (2.5 vs 4.2 days; P = 0.03). In general, partial replacement of waste milk with TM (2 l/day) may be recommended to feed dairy calves at an early stage of life to support a higher growth rate and health benefits.     

Use of inhibitors to identify essential cysteine proteinases of Trichomonas vaginalis

Designing cysteine proteinase inhibitors as antitrichomonal drugs requires knowledge of which cysteine proteinases are essential to the parasite. In an attempt to obtain such information, the effects of a number of cysteine proteinase inhibitors on trichomonad growth in vitro and proteinase activity were investigated. The broad specificity inhibitor trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane (known as E-64) had little effect on growth of Trichomonas vaginalis (27% inhibition at 280 μM, none at 28 μM) even though the addition of 2.8 μM E-64 to growth medium resulted in inhibition of all but two (apparent molecular masses: 35 k and 49 k) of the parasite's proteinases detected by gelatin SDS-PAGE. This shows that many of the parasite's cysteine proteinases are not essential for growth in axenic culture. In contrast, a peptidyl acyloxymethyl ketone, N-benzoyloxycarbonyl-Phe-Ala-CH₂OCO-(2,6,-(CF₃)₂)Ph, at 16 μM killed T. vaginalis and severely inhibited growth of Tritrichomonas foetus. Exposure of Trichomonas vaginalis to 16 μM of this compound for 1 h resulted in both the 35 kDa and 49 kDa proteinases being inhibited, whereas some other proteinases were unaffected. Similar distinctions between the inhibitor sensitivity of the parasite's cysteine proteinases were apparent when a biotinylated peptidyl diazomethyl ketone was used to detect active proteinases. These data suggest that the growth inhibitory effects of the peptidyl acyloxymethyl ketone are through inhibition of cysteine proteinases that are not affected when the parasites are grown in the presence of E-64. At least one of these enzymes, which include the 35 kDa and 49 kDa cysteine proteinases, must be essential and so a suitable target for chemotherapeutic attack.     

Capsicum annuum proteinase inhibitor ingestion negatively impacts the growth of sorghum pest Chilo partellus and promotes differential protease expression

BackgroundChilo partellus is an important insect pest infesting sorghum and maize. The larvae internalize in the stem, rendering difficulties in pest management. We investigated the effects of Capsicum annuum proteinase inhibitors (CanPIs) on C. partellus larvae by in-vitro and in-vivo experiments.MethodsRecombinant CanPI-7 (with four-Inhibitory Repeat Domains, IRDs), -22 (two-IRDs) and insect proteinase activities were estimated by proteinase assays, dot blot assays and in gel activity assays. Feeding bioassays of lab reared C. partellus with CanPI-7 and -22 were performed. C. partellus proteinase gene expression was done by RT-PCR. In-silico structure prediction of proteinases and CanPI IRDs was carried out, their validation and molecular docking was done for estimating the interaction strength.ResultsLarval proteinases of C. partellus showed higher activity at alkaline pH and expressed few proteinase isoforms. Both CanPIs showed strong inhibition of C. partellus larval proteinases. Feeding bioassays of C. partellus with CanPIs revealed a dose dependent retardation of larval growth, reduction of pupal mass and fecundity, while larval and pupal periods increased significantly. Ingestion of CanPIs resulted in differential up-regulation of C. partellus proteinase isoforms, which were sensitive to CanPI-7 but were insensitive to CanPI-22. In-silico interaction studies indicated the strong interaction of IRD-9 (of CanPI-22) with Chilo proteinases tested.ConclusionsOf the two PIs tested, CanPI-7 prevents induction of inhibitor insensitive proteinases in C. partellus so it can be explored for developing C. partellus tolerance in sorghum.General significanceIngestion of CanPIs, effectively retards C. partellus growth; while differentially regulating the proteinases.