Safety and efficacy of a mesenchymal stem cell intramammary therapy in dairy cows with experimentally induced Staphylococcus aureus clinical mastitis.

Although, antibiotics are effective in the treatment of bovine mastitis, they do not address the regeneration of mammary glandular tissue and have been associated to the increment in antimicrobial resistance worldwide. Considering the necessity of alternative therapies for this disease of high economic impact and the reported regenerative and antibacterial effects of mesenchymal stem cell (MSCs), we evaluated the safety and efficacy of an allogenic MSC-based intramammary therapy in dairy cows with experimentally induced Staphylococcus aureus clinical mastitis. In a safety trial, heifers were inoculated intramammarily with a 2.5 × 107-suspension of bovine fetal AT-MSCs on experimental days 1 and 10. Animals were evaluated clinically on a daily basis during a 20-day experimental period and blood samples were collected for hemogram determination and peripheral blood leukocytes (PBLs) isolation. In an efficacy trial, Holstein Friesian cows were inoculated with S. aureus and treated intramammarily with vehicle (NEG; days 4 and 10), antibiotics (ATB; days 4 and 5) or a suspension of 2.5 × 107 AT-MSCs (MSC; days 4 and 5). Cows were clinically evaluated daily and milk samples were collected for somatic cell count (SCC) and colony forming units (CFU). Blood samples were collected for serum haptoglobin and amyloid A determination. Intramammary administration of two doses of bovine fetal AT-MSCs in healthy cows did not induce changes in clinical or hematological variables, and gene expression profiles in PBLs associated to activation (CD4, CD8, CD25, CD62L and CD69) and proinflammatory cytokines (CCL2, CCL5, IL2, CXCL3, IFNγ, and TNFα). Quarters of MSC group of cows had similar SCC log/mL in milk compared to infected quarters of ATB or NEG cows. However, quarters of MSC cows had lower CFU log/mL in milk compared to quarters of NEG cows. Intramammarily inoculation of repeated doses of 2.5 × 107 allogenic AT-MSCs did not induce clinical or immunological response in healthy cows. Moreover, MSC-intramammary treatment reduced bacterial count in milk of cows with S. aureus clinical mastitis compared to untreated cows. This work provides initial evidence for the safety and efficacy of an allogenic MSC-based intramammary therapy for the treatment of bovine mastitis.

Retrospective evaluation of milk production and culling risk following either surgical, toggle-pin suture or conservative treatment of left displaced abomasum in Chilean dairy cows.

AIMS To describe milk yield and culling risk in cows diagnosed with left displacement of abomasum (LDA) treated either conservatively, by right flank pyloric omentopexy, or rolling and toggling, compared with normal herdmates from four Chilean dairy herds. METHODS Historical records were obtained from four commercial dairy farms located in Central Chile for cows with a history of LDA between 2010 and 2012, and healthy herdmates. Cows with LDA were categorised into three groups: cows treated with right omentopexy (ST, n=58), cows treated by toggle suturing (TT, n=15) and cows treated conservatively (CT, n=56). Control cows (n=129) were selected from unaffected cows, matched by days in milk (DIM), parity and herd with affected cows. Groups were compared for risk of culling up to 300 DIM and for milk production up to 5 months of lactation using survival and Cox proportional hazard models and mixed models for repeated measures, respectively. RESULTS Compared with cows in the Control group, the risk of being culled up to 300 DIM was 9.1 (SE 0.62) times greater in ST cows, 10.4 (SE 0.68) times greater in TT cows, and 37.3 (SE 0.61) times greater in CT cows (p<0.01). In the first 5 months of lactation, compared with cows in the Control group, mean daily milk production was 23.3 (SE 1.5) kg less in ST cows, 15.3 (SE 1.6) kg less in TT cows, and 30.1 (SE 1.3) kg less in CT cows (p<0.001). CONCLUSIONS AND CLINICAL RELEVANCE Cows in four dairy herds in central Chile diagnosed and treated for LDA produced significantly less milk and had a higher risk of culling than healthy herdmates. Although cows treated surgically or with toggle suture never recovered to the extent of healthy cows, they produced more milk than cows treated conservatively. However, the retrospective nature of the data, the inclusion of only four herds and the non-random allocation to treatments means that these conclusions cannot be extrapolated to the overall dairy cattle population in Chile.

Relationship between serum nonesterified fatty acids at calving and the incidence of periparturient diseases in Holstein dairy cows.

The objective was to describe the relationship between concentration of serum nonesterified fatty acids (NEFAs) at calving and the incidence of periparturient disorders in Chilean Holstein dairy cows (Bos taurus). The study was conducted at two dairies (central Chile) with 700 milking cows each and similar management. Between July 2006 and March 2007, 350 cows were selected, and concentrations of serum NEFAs were determined at calving. The incidence of milk fever (MF), retained fetal membranes (RFMs), metritis, and clinical mastitis from calving to 100 d in lactation were consistently recorded. The relationship between concentration of serum NEFAs at calving and the incidence of periparturient diseases was determined using logistic regression. The main explanatory variable was concentration of serum NEFAs at calving. The incidence of MF, RFM, metritis, and mastitis was 5.4%, 15.6%, 10.8%, and 14.4%, respectively. There was no association between concentration of NEFAs at calving and the incidence of these conditions when the median value of NEFAs (0.9 mEq/L) was used as a cutoff. However, when the 75th percentile (1.2 mEq/L) was used as the cutoff, cows with values <1.2 mEq/L were 0.45 and 0.32 times as likely to develop clinical mastitis and MF, respectively, compared with cows with values >or=1.2 mEq/L. When the 90th percentile (1.6 mEq/L) was used as a cutoff, cows with values <1.6 mEq/L were 0.25 times as likely to develop clinical mastitis compared with cows with values >or=1.6 mEq/L. As a continuous variable, for every 0.1 mEq/L increment in NEFAs at calving, cows were 1.11 times more likely to experience clinical mastitis. In conclusion, cows with NEFA concentrations >or=1.2 mEq/L had a higher incidence of clinical mastitis and MF than that of cows with values <1.2 mEq/L.

Characterization of estrus detection, conception and pregnancy risk of Holstein cattle from the central area of Chile.

The objectives were to characterize the estrus detection risk (HDR), conception risk (CR), and pregnancy rate (PR) of postpartum (pp) Holstein cattle from the central area of Chile. The study used records of 2269 lactations from six dairy farms in central Chile (Mediterranean-type climate) during 2004. Three 21-d periods for estrus detection were considered (50-70, 71-91, and 92-112d pp). Estrus detection risk, CR, and PR at the first, second, and third periods were analyzed by logistic regression, whereas overall PR at the end of the 63-d study (112d pp) was assessed with survival analysis. The overall HDR was 51.1%. The HDR, CR, and PR were 48.4, 42.2, and 17.3%, respectively, during the first period; 52.8, 41.8, and 20.5% during the second period; and 52.9, 39.2, and 19.7% during the third period. The HDR was lower during Period 1 than during Periods 2 and 3 (P< or =0.05). Conception risks were not different among periods (P>0.05); however, PR was lower during Period 1 than during Periods 2 and 3 (P< or =0.05). Overall PR over time differed among parities, but was not significantly different among seasons. There were no significant interactions among parity, season and herd for HDR, CR and PR for the three 21-d periods. Parity 1 had higher CR and PR than Parity 2 and 3+ during Period 3. Overall, survival curves for the risk of non-pregnancy among parities (1, 2, 3 or greater) were different over time (P< or =0.05). Cows of Parity 1 became pregnant earlier than cows of Parity 2, and Parity 3 or greater. Survival curves for the risk of non-pregnancy among seasons (summer, fall, winter, and spring) were not different over time (P>0.05).

Histological characteristics and steroid concentration of ovarian follicles at different stages of development in pregnant and non-pregnant dairy cows.

The aim of the study was to investigate the histological characteristics and steroid concentrations in follicular fluid of different populations of follicles at different stages of development, during pregnancy and the oestrous cycle in cows. Follicles from ovaries collected at a slaughterhouse were allocated into three size categories (small, 2-5.9 mm; medium, 6-13.9 mm; and large, 14-20 mm) in pregnant and non-pregnant cows. Slices were stained with HE and PAS for histological analysis. Follicular fluid was pooled according to size and pregnancy status and estradiol, testosterone and progesterone concentrations in follicular fluid were determined by RIA. Characteristics of healthy follicles did not differ, regardless of follicle size or pregnancy status. Total histological atresia was significantly higher in pregnant cows than in non-pregnant cows (p < 0.05). Estradiol increased and testosterone decreased significantly, while follicles increased in size, in both non-pregnant and pregnant cows (p < 0.05). Nonpregnant cows had the highest estradiol values in follicles of all sizes. Medium and large follicles from pregnant cows showed the lowest testosterone concentration (p < 0.05). Progesterone levels increased with follicle size only in non-pregnant animals. In large follicles, progesterone concentration was significantly higher in non-pregnant cows than in pregnant cows (p < 0.05). Considering steroid concentration and histological findings, most large follicles might be atretic during pregnancy in cattle.

Ultrastructure of bovine ovarian follicles induced to extended growth by perioestrous suprabasal progesterone levels.

The present study was undertaken to determine if a short-term prolonged growth of the ovulatory follicle (12 to 18 h after expected time of ovulation), induced by progesterone implants, would cause ultrastructural changes in the follicular wall. Oestrous behaviour, follicular growth, follicular and blood plasma levels of oestradiol-17 beta, progesterone and plasma luteinizing hormone (LH) were monitored in heifers oophorectomized at 9 to 12 h (controls) or 36 h after the onset of oestrus, in order to sample the pre-ovulatory follicle present. The suprabasal plasma progesterone concentrations (approximately 1.2 nmol L-1) allowed expression of oestrus at the expected time, but ovulation was delayed owing to the absence of a LH-surge. The resulting prolongation of follicle growth was associated with mild degenerative changes in the follicle wall, i.e. both granulosa and thecal cells presented increased electron density, higher amounts of secondary lysosomes and lipid droplets, increased intercellular spaces with presence of debris. No signs of luteinization were seen.

Effect of induced suprabasal progesterone levels around estrus on plasma concentrations of progesterone, estradiol-17beta and LH in heifers.

A controlled study was carried out to investigate the effects of suprabasal plasma progesterone concentrations on blood plasma patterns of progesterone, LH and estradiol-17beta around estrus. Heifers were assigned to receive subcutaneous silicone implants containing 2.5 g (n=4), 5 g (n=4), 6 g (n=3), 7.5 g (n=3) or 10 g (n=4) of progesterone, or implants without hormone (controls, n=5). The implants were inserted on Day 8 of the cycle (Day 0=ovulation) and left in place for 17 d. The time of ovulation was determined by ultrasound scanning. Blood was collected daily from Days 0 to 14 and at 2 to 4-h intervals from Days 15 to 27. Control heifers had the lowest progesterone concentrations on Days 20.5 to 21 (0.5 +/- 0.1 nmol L(-1)); a similar pattern was observed in heifers treated with 2.5 and 5 g of progesterone. In the same period, mean progesterone concentrations in the heifers treated with 6, 7.5 and 10 g were larger (P < 0.05) than in the controls, remaining between 1 and 2.4 nmol L(-1) until implant removal. A preovulatory estradiol increase started on Days 16.4 to 18.4 in all the animals. In the controls and in heifers treated with 2.5 and 5 g of progesterone, estradiol peaked and was followed by the onset of an LH surge. In the remaining treatments, estradiol release was prolonged and increased (P < 0.05), while the LH peak was delayed (P < 0.05) until the end of the increase in estradiol concentration. The estrous cycle was consequently extended (P < 0.05). In all heifers, onset of the LH surge occurred when progesterone reached 0.4 to 1.2 nmol L(-1). The induction of suprabasal levels of progesterone after spontaneous luteolysis caused endocrine asynchronies similar to those observed in cases of repeat breeding. It is suggested that suprabasal concentrations of progesterone around estrus may be a cause of disturbances oestrus/ovulation.

Effect of intracervical administration of a prostaglandin E2 gel in pregnant and non-pregnant heifers.

The effects of prostaglandin E2 (PGE2) on cervical opening in non-pregnant and pregnant heifers was studied, and in the pregnant animals the effect on the embryo was studied by means of ultrasonography. In four consecutive experiments, 5 ml of saline, a gel containing 2 mg or 6 mg PGE2 was administered intracervically to four non-pregnant heifers, and 2 mg of PGE2 was administered to heifers pregnant 33 to 40 days. All the groups treated with PGE2 experienced an increase in the concentrations of prostaglandin metabolite in plasma (P < 0.05) shortly after administration, which reached a peak 15 to 30 minutes after administration. An increase in cervical opening was evident in all the PGE2-treated heifers (P < 0.05) from three hours after treatment. There was no difference between the effect of the two doses. The heifers which received saline did not show any significant changes. In addition, the treated heifers showed cervical softening, congestion and mucus secretion which were more pronounced in the pregnant heifers. The embryos were not affected. Plasma progesterone concentrations remained unchanged in all the experiments.