Juhani Taponen

A Novel Mutation in the Maternally Imprinted PEG3 Domain Results in a Loss of MIMT1 Expression and Causes Abortions and Stillbirths in Cattle (Bos taurus)

Congenital malformations resulting in late abortions and stillbirths affect the economic wellbeing of producers and the welfare of cattle in breeding programs. An extremely high incidence of stillbirths of “half-sized” calves of normal karyotype and uninflated lungs was diagnosed in the progeny of the Finnish Ayrshire (Bos taurus) bull - YN51. No other visible anatomical abnormalities were apparent in the stillborn calves. We herein describe the positional identification of a 110 kb microdeletion in the maternally imprinted PEG3 domain that results in a loss of paternal MIMT1 expression and causes late term abortion and stillbirth in cattle. Using the BovineSNP50 BeadChip we performed a genome-wide half-sib linkage analysis that identified a 13.3 Mb associated region on BTA18 containing the maternally imprinted PEG3 domain. Within this cluster we found a 110 kb microdeletion that removes a part of the non-protein coding MER1 repeat containing imprinted transcript 1 gene (MIMT1). To confirm the elimination of gene expression in calves inheriting this deletion, we examined the mRNA levels of the three maternally imprinted genes within the PEG3 domain, in brain and cotyledon tissue collected from eight fetuses sired by the proband. None of the fetuses that inherited the microdeletion expressed MIMT1 in either tissue. The mutation, when inherited from the sire, is semi-lethal for his progeny with an observed mortality rate of 85%. The survival of 15% is presumably due to the incomplete silencing of maternally inherited MIMT1 alleles. We designed a PCR-based assay to confirm the existence of the microdeletion in the MIMT1 region that can be used to assist cattle breeders in preventing the stillbirths.

Induction of ovulation with gonadotropin-releasing hormone during proestrus in cattle: influence on subsequent follicular growth and luteal function

Induction of ovulation by administration of gonadotropin-releasing hormone (GnRH) is commonly practiced in cattle to treat repeat breeders or cows exhibiting long estrous periods. This treatment may, however, disturb normal reproductive functions if timing is incorrect. The objective of the present study was to investigate the effect of exogenous GnRH on estradiol secretion of the ovulatory follicle, occurrence of ovulation, development and function of the corpus luteum (CL) and growth of a dominant follicle after ovulation in the bovine, when GnRH treatment was given before the expected physiological LH-surge. Luteolysis was induced by cloprostenol (PG) in three cows and six heifers. Every animal was assigned once to each of the following treatment or control manipulations, receiving either a single dose (0.1 mg) of GnRH (gonadorelin) at (1) 24 h (T1), (2) 48 h (T2), or (3) 72 h (T3) after PG, or (4) no gonadorelin (control manipulation, C). Ovaries were scanned by ultrasound and blood samples were collected for progesterone (P4) and estradiol-17beta (E-17beta) determination. Growth curves of dominant follicles between treatment I and the control differed significantly (P < 0.01). One day after ovulation, the diameter of the dominant follicle was almost 1 mm larger in T1. This difference remained almost unchanged during the entire follow-up period. The recruitment of a new follicular wave after ovulation seemed to occur earlier. Development of CL and levels and profiles of P4-production remained unaffected. When GnRH was given 1 day after PG injection, two animals showed significantly different development of CL (P < 0.05) and of P4-production (both in concentrations [P < 0.05] and profile [P < 0.01]). After normal ovulation and CL development, luteolysis took place on days 5 or 6 after ovulation, and animals ovulated on days 9 and 10. It is suggested that early induction of ovulation with GnRH can cause shortened luteal function in cattle and, ultimately, reduced fertility.

A novel automated fluorometric assay to evaluate sperm viability and fertility in dairy bulls

The artificial insemination (AI) industry is in need of an objective and rapid, but inexpensive method to evaluate frozen thawed bull semen ejaculates. This study presents a new fluorescence method that uses an automatized fluorometer and fluorophore stain propidium iodide that stains only those cells with damaged membranes. The fluorescence of the semen sample and the totally killed subsample were measured simultaneously, and viability was calculated. Every semen batch was analyzed before use in AI. For fertility evaluation, the nonreturn rates (NR%) obtained from 92,120 inseminations with the analyzed batches were recorded from 166 bulls (436 batches). This study confirms a 3.9% better NR% for the Finnish Holstein-Friesian breed than for Finnish Ayrshire. There was a clear seasonality in NR%: it differed (5.3%) significantly, being best in summer to autumn (June to October) and lowest in winter (January to March). The fluorometer method was fast and easy. The correlation between the total number of viable spermatozoa in an insemination dose and field fertility was low but significant (r = 0.051, P = 0.016), suggesting that the plasma membrane integrity evaluation can serve as a cost-beneficial quality control method of frozen-thawed semen at bull stations.

Equol in milk of dairy cows is derived from forage legumes such as red clover

The intake of isoflavones and the resulting equol contents of both plasma and milk of the same red clover-fed cows are reported for the first time in cyclic change-over design study. Cows were fed four different red clover silages and two timothy-meadow fescue silages as controls. The red clover silages contained daidzein, formononetin, biochanin A and genistein, whereas the timothy-meadow fescue silages contained no isoflavones. We found a strong association (y = 0.071x+2.75, R 2 0.71) between the formononetin intake (x) and equol concentration (y) in the plasma, while the formononetin intake and milk equol concentration were weakly associated (y = 0.0035x+0.358, R 2 0.20). This means that a small part of the total formononetin in the silage is secreted into milk as equol. The mean equol contents in plasma and milk of cows fed red clover silage diets were in the range of 4.6-8.4 mg/l and 458-643 microg/l, respectively, while the respective values for the control diets were in the range of 0.8-1.5 mg/l and 171-287 microg/l. We showed that shorter growing periods of red clover resulted in higher silage formononetin contents and plasma and milk equol contents, suggesting that the equol content of milk can be manipulated by varying the harvesting strategy of red clover. We conclude that milk equol is derived from the formononetin of red clover silage and that milk from red clover-fed cows can be considered as a source of equol in human nutrition.

Short estrous cycles and estrous signs after premature ovulations induced with cloprostenol and gonadotropin-releasing hormone in cyclic dairy cows

The aim of the present study was to confirm earlier findings, obtained with a small number of animals, that gonadotropin-releasing hormone (GnRH) can shorten corpus luteum functional life when it is administered 24 h after cloprostenol (PG) treatments given 7-9 days after estrus. In addition, the effects of two treatments, PG alone or PG + GnRH given before mid-diestrus, on signs of estrus were studied. Sixty cows in farm conditions were used in the experiment. Eight days after natural estrus, they were given an intramuscularly (i.m.) treatment of cloprostenol (0.5 mg). The animals were then divided into two groups. One group (n = 25) received an i.m. treatment of gonadorelin (0.1 mg) 24 h after the PG treatment (PG + GnRH group), while another group (n = 35) served as controls without any further treatment (PG group). Estrous signs were recorded. Progesterone concentrations were measured from samples of whole milk. No short cycles were observed in the PG group, whereas 33% of the cows in the PG + GnRH group exhibited premature luteal regression (P < 0.05). Cloprostenol treatment on Day 8 had no effect on the intensity of the estrous signs. Instead, GnRH treatment 24 h after PG treatment weakened the estrous signs significantly (P < 0.01). It is concluded that GnRH administration 24 h after a PG treatment given 8 days after estrus can cause short estrous cycles in some cows on an individual basis.

Premature prostaglandin F2α secretion causes luteal regression in GnRH-induced short estrous cycles in cyclic dairy heifers

This study aimed to confirm that the luteolysis in normal-cycling dairy heifers seen during short estrous cycles induced with cloprostenol (Clp) and GnRH administered 24h apart is caused by a premature release of prostaglandin F(2alpha) (PGF(2alpha)). A further aim was to study the PGF(2alpha) release pattern more closely to determine whether it resembles the spontaneous release occurring during normal regression of the corpus luteum (CL) or whether PGF(2alpha) is continuously secreted after the induced ovulations, leading to short estrous cycles. Twenty-four Ayrshire heifers were allotted to four equally sized groups. After estrus synchronization with 0.5mg of Clp, a new luteolysis was induced with 0.5mg of Clp on Day 6 (groups T-d6 and C-d6) or Day 7 (groups T-d7 and C-d7) after ovulation. Gonadorelin (0.1mg i.m.) was given to groups T-d6 and T-d7 to induce premature ovulation 24h later. Groups C-d6 and C-d7 served as controls. Ovaries were examined daily by transrectal ultrasonography, while blood samples (for progesterone and 15-ketodihydro-PGF(2alpha) analyses) were obtained via a jugular catheter every 3h, starting from the second Clp treatment and continuing for 9 days postovulation. Unresponsiveness to Clp or anovulation resulted in 4 C-d6 heifers being excluded. Four heifers in group T-d6 and three in group T-d7 had a short estrous cycle of 8-12 days, while all others had a cycle of normal length. Significant elevations in 15-ketodihydro-PGF(2alpha) concentrations with recurrent high peaks coincided with a decrease in progesterone concentration and were detected in all heifers that showed a short estrous cycle, but not in any heifers with normal estrous cycles in groups T and C. In conclusion, a premature release of PGF(2alpha), which closely resembles its release during spontaneous luteolysis, causes luteal regression in these short cycles.

Seroprevalence of canine herpesvirus-1 and Brucella canis in Finnish breeding kennels with and without reproductive problems.

We compared the serological status of Brucella canis and canine herpesvirus-1 (CHV-1) in Finnish breeding kennels with and without reproductive problems. Dogs from kennels with reproductive problems had significantly higher CHV-1 titres than dogs from kennels having no reproductive problems (p < 0.001). In dogs from kennels with reproductive problems 100% (32/32) had positive titres, whereas in dogs from kennels without reproductive problems 65% (22/34) had positive titres. The median titre for dogs from kennels with reproductive problems was 1 : 160 and for dogs from kennels without reproductive problems 1 : 80. The high prevalence of positive CHV-1 titres in this study indicates that prevention of the disease is difficult and reinforces the need to minimize the reproductive problems caused by CHV-1. All 388 dogs from 94 kennels had negative B. canis titres.

Effects of abomasal infusion of tallow or camelina oil on responses to glucose and insulin in dairy cows during late pregnancy.

Late pregnancy is associated with moderate insulin resistance in ruminants. Reduced suppression of lipolysis by insulin facilitates mobilization of nonesterified fatty acids (NEFA) from adipose tissue, resulting in elevated plasma NEFA concentrations. Decrease in dry matter intake (DMI) before parturition leads to accelerated lipomobilization and increases plasma NEFA, which may further impair insulin sensitivity. The aim of the study was to evaluate the effects of elevation of plasma NEFA concentration by abomasal infusions tallow (TAL) or camelina oil (CAM) on whole-body responses to exogenous glucose and insulin. We further assessed whether CAM, rich in C18:3n-3, enhances whole-body insulin sensitivity compared with TAL. Six late-pregnant, second-parity, rumen-cannulated dry Ayrshire dairy cows fed grass silage to meet 95% of metabolizable energy requirements were used in a replicated 3 × 3 Latin square with 5-d periods and 5 recovery days between each period. Treatments consisted of abomasal infusion of 500 mL/d (430 g of lipids/d) of water (control), TAL, or CAM administered in 10 equal doses daily. Intravenous glucose tolerance test (IVGTT) and i.v. insulin challenge (IC) were performed on d 5 after 98 and 108 h of treatment infusions, respectively. Infusion of lipids increased basal plasma NEFA concentrations on d 5 (CAM: 0.25; TAL: 0.28; control: 0.17 mmol/L). Following glucose injection, the rate of glucose clearance (CR) was lower in lipid-treated cows (CAM: 1.34; TAL: 1.48; control: 1.74%/min) and time to reach half-maximal glucose concentration (T(1/2)) was longer (CAM: 54; TAL: 47; control: 42 min). Similar responses were observed after insulin injection. Increased plasma NEFA concentration tended to decrease insulin secretion in IVGTT. Infusion of CAM increased plasma C18:3n-3 content (CAM: 26.4; TAL: 16.1; control: 20.9 g/100g of fatty acids). Data suggest that CAM had an insulin-sensitizing effect, because the disposition index and insulin sensitivity index, derived from minimal model analysis, were higher in CAM than in TAL during IVGTT, and lower insulin concentrations during IC led to similar glucose clearance in CAM as in TAL. These results indicate that elevated plasma NEFA concentration per se induces whole-body insulin resistance in late-pregnant dry cows.

Ectopic KIT Copy Number Variation Underlies Impaired Migration of Primordial Germ Cells Associated with Gonadal Hypoplasia in Cattle (Bos taurus)

Impaired migration of primordial germ cells during embryonic development causes hereditary gonadal hypoplasia in both sexes of Northern Finncattle and Swedish Mountain cattle. The affected gonads exhibit a lack of or, in rare cases, a reduced number of germ cells. Most affected animals present left-sided gonadal hypoplasia. However, right-sided and bilateral cases are also found. This type of gonadal hypoplasia prevails in animals with white coat colour. Previous studies indicated that gonadal hypoplasia is inherited in an autosomal recessive fashion with incomplete penetrance. In order to identify genetic regions underlying gonadal hypoplasia, a genome-wide association study (GWAS) and a copy number variation (CNV) analysis were performed with 94 animals, including 21 affected animals, using bovine 777,962 SNP arrays. The GWAS and CNV results revealed two significantly associated regions on bovine chromosomes (BTA) 29 and 6, respectively (P=2.19 x 10-13 and P=5.65 x 10-6). Subsequent cytogenetic and PCR analyses demonstrated that homozygosity of a ~500 kb chromosomal segment translocated from BTA6 to BTA29 (Cs29 allele) is the underlying genetic mechanism responsible for gonadal hypoplasia. The duplicated segment includes the KIT gene that is known to regulate the migration of germ cells and precursors of melanocytes. This duplication is also one of the two translocations associated with colour sidedness in various cattle breeds.

Treating cattle with antibiotics affects greenhouse gas emissions, and microbiota in dung and dung beetles

Antibiotics are routinely used to improve livestock health and growth. However, this practice may have unintended environmental impacts mediated by interactions among the wide range of micro- and macroorganisms found in agroecosystems. For example, antibiotics may alter microbial emissions of greenhouse gases by affecting livestock gut microbiota. Furthermore, antibiotics may affect the microbiota of non-target animals that rely on dung, such as dung beetles, and the ecosystem services they provide. To examine these interactions, we treated cattle with a commonly used broad-spectrum antibiotic and assessed downstream effects on microbiota in dung and dung beetles, greenhouse gas fluxes from dung, and beetle size, survival and reproduction. We found that antibiotic treatment restructured microbiota in dung beetles, which harboured a microbial community distinct from those in the dung they were consuming. The antibiotic effect on beetle microbiota was not associated with smaller size or lower numbers. Unexpectedly, antibiotic treatment raised methane fluxes from dung, possibly by altering the interactions between methanogenic archaea and bacteria in rumen and dung environments. Our findings that antibiotics restructure dung beetle microbiota and modify greenhouse gas emissions from dung indicate that antibiotic treatment may have unintended, cascading ecological effects that extend beyond the target animal.