Reproducibility of endometrial cytology using cytobrush technique and agreement for the diagnosis of subclinical endometritis between five predefined endometrial sites

The objectives of this study were as follows: (a) to assess the reproducibility of polymorphonuclear neutrophil (PMN) cell counts at five predefined endometrial sites (corpus uteri, left horn base, right horn base, left horn tip and right horn tip) and (b) to determine the agreement for the diagnosis of subclinical endometritis (SE) between the different endometrial sites. Forty milking cows between 28 and 34 days post‐partum were enrolled for endometrial sampling using cytobrush technique. Intraclass correlation coefficient (ICC) was calculated to evaluate the reproducibility of PMN counts at different sites. The right horn base was found to have the greatest agreement of PMN counts with the other endometrial sites (ICC = 0.66–0.85). Twenty‐eight of 40 cows showed no signs of clinical endometritis and were used for evaluation of agreement for the diagnosis of SE, analysed by using Cohen´s kappa (κ) statistics. Agreement for SE diagnosis with PMN cut‐off ≥5% was greatest between the right horn base and the right horn tip (κ = 0.84), and with PMN cut‐off ≥18% between the right horn base and left horn tip (κ = 1.0), respectively. The results indicate that the right horn base can be regarded as suitable for cytobrush sampling. The probability to detect an animal positive for SE (PMN ≥ 5%) with a single cytobrush sampling was 51.0%; thus, a second sampling is recommended to improve the accuracy.     

Changes of leukocyte counts and expression of pro‐ and anti‐inflammatory cytokines in peripheral leukocytes in periparturient dairy cows with retained fetal membranes

In dairy cows, retained fetal membranes (RFM) affect reproductive performance. The aim of this study was to examine the leukocyte counts and the gene expression of tumour necrosis factor α (TNFα), interleukin 1β (IL‐1β), IL‐8, and IL‐10 in polymorphonuclear leukocytes (PMNs) and peripheral blood mononuclear cells (PBMCs) in cows with (n = 5) or without (n = 5) RFM during the peripartum period. The lymphocyte counts in RFM cows were higher than those in control cows throughout the experiment (p < .05). The expression of IL‐8 in PMNs of control cows was higher (p < .05) compared with that of RFM cows postpartum. In cows with RFM, IL‐1β expression was higher (p < .05) in PMNs at 6 weeks postpartum whereas the expression of IL‐1β was lower (p < .05) in PBMCs at 4 weeks postpartum. The expression of IL‐10 in PBMCs of control cows was higher (p < .05) than that of RFM cows at 2 weeks prepartum and 4 weeks postpartum. Taken together, our data indicate that changes of gene expression of pro‐ and anti‐inflammatory cytokines in RFM cows might be associated with the delayed placental separation and development of uterine inflammation in RFM cows.     

A progestin-based treatment with a high dose of estradiol benzoate normalizes cyclic changes in endometrial EGF concentrations and restores fertility in repeat breeder cows

The objective of the present study was to examine the efficacy of a progestin-based treatment with a high dose of estradiol benzoate (EB) to normalize the epidermal growth factor (EGF) profile in the uterine endometrium and restore fertility in repeat breeder cows. Repeat breeder cows without peaks in their endometrial EGF concentrations on Days 3 and 14 of the estrous cycle were used throughout the study. The effect of 1 (standard dose), 2.5 and 5 mg of EB in a progestin-based treatment protocol (EB1, EB2.5 and EB5 treatments, respectively; n=5 for each group) on endometrial EGF concentrations was first examined. The EB1 and EB2.5 treatments in the repeat breeder cows produced a suppressed response in endometrial EGF compared with EB1 treatment in the fertile controls (n=5) and failed to restore the normal EGF profile during the next estrous cycle. However, EB5 treatment produced an increase in EGF concentrations similar to the fertile controls and normalized the endometrial EGF profile. The effects of the EB1 and EB5 treatments (n=30 for each group) on the endometrial EGF profile and fertility were then examined in the repeat breeder cows. The proportion of cows, with an EGF profile normalized by the treatments was higher in the EB5 group (66.7%) than in the EB1 (30.0%) and untreated control (13.3%; n=30) groups (P<0.01). The pregnancy rates of the cows having a normal EGF profile after treatment in the EB1 and EB5 groups were similar (88.9 and 85.0%, respectively) and higher than those of the cows having an abnormal profile within the same groups (19.0 and 30.0%, respectively, P<0.01). In summary, the endometrial response to EB in terms of the EGF concentration was suppressed in repeat breeder cows. A high dose (5 mg) of EB in a progestin-based treatment was found to be effective for restoration of a normal EGF profile and fertility in repeat breeder cows having lesser endometrial EGF concentrations on Days 3 and 14.     

A Study of Endometritis Causing Repeat Breeding of Cycling Iraqi Buffalo Cows

The objectives of this study were to determine the non‐specific aerobic and anaerobic bacterial causes of endometritis causing repeat breeding of cycling Iraqi buffalo cows at Nineveh province, validate diagnostic criteria for endometritis and to evaluate the treatment efficiency of using systemic or intra‐uterine infusion of antibiotics for the treatment of endometritis. Data were collected from 60 buffalo cows with history of repeat breeding in different herds. All buffaloes were subjected to detailed clinical examination including external inspection, vaginoscopy and transrectal palpation of the cervix, uterus and ovaries. Swabs for bacteriology and biopsies for histopathology were collected from the uterine lumen from each cow. Character, odour and estimation of polymorphonuclear cells (PMN) of the vaginal mucus were scored. Blood samples were collected from cows for creatine kinase (CK) and aspartate aminotransferase (AST) measurement. Treatment conducted using oxytetracycline with tylosin in local intrauterine infusion or systemically with hormonal treatment. The most pre‐disposing factor for uterine infection was retained placenta (13.3%). The most prevalent bacteria in uterine lumen were E. coli (23%), Archanobacterium pyogenes (13%) and Staphylococcus aureus (10%) were mostly isolated from buffaloes with repeat breeding. Vaginal mucus character score was associated with the bacterial growth density score. The difference in PMN was highly significant (p < 0.01) in animals with repeat breeding than control groups. In addition, PMNs was significantly (p < 0.01) correlated r = 0.894 with the character of vaginal discharge. High level of PMNs observed in buffaloes infected with A. pyogenes. Buffalo cows with endometritis had higher CK (321.47 ± 39.06 vs 162.01 ± 16.41 U/l) and AST (133.93 ± 12.43 vs 97.01 ± 6.86 U/l) activities (p < 0.05) than control‐heifers, but no significant difference was observed between buffalo cows with endometritis in CK (321.47 ± 39.06 vs 208.33 ± 5.84) and AST (133.93 ± 12.43 vs 156.17 ± 9.65) activities than control‐pluriparious. It could be concluded that A. pyogenes was the only non‐specific uterine pathogen directly associated with severe endometrial lesions. Vaginoscopy examination combined with palpation of uterus increase the accuracy of diagnosing endometritis and cytogenic examination of uterine discharge is more reliable method of establishing the presence or absence of uterine inflammation in buffalo cows. Animals with repeat breeding (endometritis) showed clinical cure and improved pregnancy in all treatment groups with no significant difference. The use of oestradiol in repeat breeder cases has no effect in improving neither clinical cure rate nor pregnancy rate.     

Inflammatory cell infiltration as an indicator of Staphylococcus aureus infection and therapeutic efficacy in experimental mouse mastitis

Staphylococcus aureus intramammary colonization of the mouse mammary gland induces migration of polymorphonuclear neutrophils (PMNs) similar to that observed during bovine mastitis. In the present study, a method combining acridine orange staining, fluorescence microscopy and computer-assisted image analysis has been developed to quantitate PMN infiltration in a mouse model of mastitis. This was carried out using paraffin embedded sections, and using this method, we showed that the presence of PMNs increased with the number of bacteria present in tissues. Nearly 400 and 1100 times more PMNs were counted in the mammary gland tissue after 12 and 24 h of infection, respectively, compared to mice infected for 6 h. Treatment with the antibiotic cephapirin at 10 or 25 mg/kg reduced PMN infiltration by 71 and 85%, respectively. In conclusion, this method can be used to quantitate PMN infiltration as a marker of inflammation and bacterial burden in infected tissue sections.     

Relationships between endometrial cytology and interval to first ovulation, and pregnancy in postpartum dairy cows in a single herd

The objectives were to investigate the relationships between endometrial cytology (EC) and interval from calving to first ovulation, and pregnancy in dairy cows, and that between uterine fluid and EC. On day 25 postpartum, 39 dairy cows were grouped based on EC, as having low (?8%) or high (>8%) polymorphonuclear cells (PMN), and the quantity of uterine fluid was assessed by ultrasound. The interval from calving to first ovulation was shorter in low, than in high PMN cows (32 vs. 45 d). A greater proportion of cows with uterine fluid had high PMN (64% vs. 21%), and the PMN increased from 14% to 34% as the quantity of uterine fluid increased. The mean interval from calving to ovulation was longer in primiparous cows with high PMN (49 d) compared to that of primiparous and multiparous cows with low PMN (28 and 29 d, respectively). Although the conception rate to first service at 92 d postpartum was not different between PMN groups, the cumulative pregnancy at 270 d tended to be higher in low than in high PMN (80% vs. 58%) multiparous cows. Also, cows that had uterine fluid on day 25 postpartum had a shorter interval from calving to pregnancy than those with no uterine fluid (161 vs. 208 d). In conclusion, combining transrectal ultrasonography with endometrial cytology on day 25 postpartum has diagnostic value in the assessment of uterine inflammation.     

Long‐term impact of puerperal metritis on the profiles of peripheral blood leukocytes in peripartum dairy cows

To determine the effects of puerperal metritis on the immune response, changes in the differential peripheral blood leukocyte counts were analyzed during the peripartum period in cows with or without metritis. Multiparous Holstein cows were examined for uterine health disorders and classified into two groups: healthy (n = 11) or metritis (n = 5) cows. The lymphocyte and monocyte counts and the proportion of CD8⁺ lymphocytes were higher in cows with metritis compared to healthy cows. Moreover, the effects of puerperal metritis on the lymphocyte counts and CD4⁺/CD8⁺ ratio persisted weeks after the uterine inflammation had self‐resolved. Taken together, the findings of the present study indicate the possible long‐term alterations of systemic immune responses in cows with puerperal uterine inflammation. © 2015 Japanese Society of Animal Science     

Relationships between endometrial cytology and interval to first ovulation,and pregnancy in postpartum dairy cows in a single herd

The objectives were to investigate the relationships between endometrial cytology (EC) and interval from calving to first ovulation, and pregnancy in dairy cows, and that between uterine fluid and EC. On day 25 postpartum, 39 dairy cows were grouped based on EC, as having low (⩽8%) or high (>8%) polymorphonuclear cells (PMN), and the quantity of uterine fluid was assessed by ultrasound. The interval from calving to first ovulation was shorter in low, than in high PMN cows (32 vs. 45 d). A greater proportion of cows with uterine fluid had high PMN (64% vs. 21%), and the PMN increased from 14% to 34% as the quantity of uterine fluid increased. The mean interval from calving to ovulation was longer in primiparous cows with high PMN (49 d) compared to that of primiparous and multiparous cows with low PMN (28 and 29 d, respectively). Although the conception rate to first service at 92 d postpartum was not different between PMN groups, the cumulative pregnancy at 270 d tended to be higher in low than in high PMN (80% vs. 58%) multiparous cows. Also, cows that had uterine fluid on day 25 postpartum had a shorter interval from calving to pregnancy than those with no uterine fluid (161 vs. 208 d). In conclusion, combining transrectal ultrasonography with endometrial cytology on day 25 postpartum has diagnostic value in the assessment of uterine inflammation.     

Degenerative Endometrial Changes do not Change the Functional Capacity of Immigrating Uterine Neutrophils in Mares

An endometritis model was used to investigate the influence of degenerative endometrial changes (endometrosis) on functional parameters of uterine neutrophils in the horse. Six hours after intrauterine application of recombinant human interleukin‐8 (rhIL‐8), the uteri of 15 mares were flushed with phosphate‐buffered saline. Quantitative and qualitative flow cytometric assays were then made to determine the absolute numbers, viability, phenotype, generation of reactive oxygen species (ROS), and phagocytic activity of immigrated polymorphonuclear neutrophilic granulocytes (PMN). Recombinant hIL‐8 attracted similarly high numbers of similarly viable PMN into the uteri of mares with or without degenerative endometrial changes. Compared with blood PMN, immigrated uterine neutrophils displayed significantly upregulated expression of CD11a/CD18 (LFA‐1) on uterine PMN whereas major histocompatibility complex class I molecules were expressed at lower densities. The ability to phagocytose opsonized streptococci did not differ between uterine and blood PMN. However, uterine PMN displayed a higher capacity to generate ROS. On average, uterine PMN of mares with degenerative endometrial changes showed phenotypical and functional characteristics similar to those of mares with a histologically healthy endometrium. Therefore, degenerative endometrial changes per se did not reduce the functional capacity of equine uterine neutrophils in mares.     

Association of intrauterine presence of Lactobacillus spp. with inflammation and pathogenic bacteria in the uterus in postpartum dairy cows

The aim of this study was to clarify the influence of Lactobacillus spp. on the degree of endometrial inflammation in the postpartum period and the relationship between Lactobacillus spp. and pathogenic bacteria in the endometrium of postpartum dairy cows. Endometrial samples were collected from 41 Holstein-Friesian cows at 4 and 8 weeks postpartum using cytobrushes for polymorphonuclear neutrophil (PMN) count and bacterial culture to isolate Lactobacillus spp., Escherichiacoli, and Trueperella pyogenes. The 4-week samples were divided into four groups (E+L+), (E+L−), (E−L+), (E−L−) according to whether endometritis was diagnosed (E+) and Lactobacillus spp. was isolated (L+). The diagnostic criterion for cytological endometritis was > 18% PMN. The average PMN% in the E+L+ group was lower than that in the E+L-group (P < 0.05) at 8 weeks postpartum. There were no significant correlations between the number of colonies of Lactobacillus spp. and E. coli or between that of Lactobacillus spp. and T. pyogenes. Lactobacillus spp. could reduce PMN% in dairy cows with endometritis during the puerperal period. In conclusion, the intrauterine presence of Lactobacillus spp. may have a positive effect on uterine involution in postpartum dairy cows.     

大肠杆菌与金黄色葡萄球菌对奶牛子宫内膜组织细胞因子表达及损伤程度的影响

本试验旨在研究体外感染金黄色葡萄球菌与大肠杆菌对奶牛子宫内膜组织中细胞因子白介素-6（IL-6）、IL-1β和IL-8的表达及损伤程度的影响。以体外培养的奶牛子宫内膜组织作为研究对象,采用1×10⁵~1×10⁹ CFU/mL大肠杆菌与金黄色葡萄球菌对奶牛子宫内膜组织进行体外感染,通过实时荧光定量PCR和ELISA方法检测两种细菌刺激后奶牛子宫内膜组织中IL-6、IL-1β及IL-8 mRNA与蛋白的表达量,并用HE染色法观察两种细菌感染后奶牛子宫内膜组织病理学切片。结果显示,1×10⁵~1×10⁹ CFU/mL大肠杆菌体外感染后,奶牛子宫内膜组织中IL-6、IL-1β和IL-8 mRNA表达量均极显著高于空白对照组（P<0.01）;金黄色葡萄球菌感染浓度为1×10⁵、1×10⁶ CFU/mL时,IL-6、IL-1β mRNA表达量极显著高于空白对照组（P<0.01）,感染浓度为1×10⁷ CFU/mL时,IL-6、IL-1β mRNA表达量显著高于空白对照组（P<0.05）,感染浓度为1×10⁶ CFU/mL时,IL-8 mRNA表达量显著高于空白对照组（P<0.05）,其他感染浓度均与空白对照组无显著差异（P>0.05）。奶牛子宫内膜组织感染1×10⁵~1×10⁹ CFU/mL金黄色葡萄球菌和大肠杆菌时,IL-6、IL-1β及IL-8蛋白表达量均极显著高于空白对照组（P<0.01）。相同浓度的大肠杆菌感染奶牛子宫内膜组织后,IL-6、IL-1β及IL-8 mRNA与蛋白表达量均极显著高于金黄色葡萄球菌感染组（P<0.01）。HE切片染色结果显示,大肠杆菌感染后仍有部分上皮细胞保留,而金黄色葡萄球菌感染后上皮细胞全部脱落。本试验结果表明,大肠杆菌与金黄色葡萄球菌感染奶牛子宫内膜组织后,引起的炎症反应不同。大肠杆菌感染后,促炎性细胞因子被显著上调,而金黄色葡萄球菌感染后破坏子宫内膜上皮细胞程度更加严重。     

Effect of prostaglandin F2α administration on uterine polymorphonuclear neutrophil counts in Japanese heavy draft horses

The objective of this study was to examine the effect of prostaglandin F2_α (dinoprost) and oxytocin administration on uterine polymorphonuclear neutrophil counts in the Japanese heavy draft mare. To compare polymorphonuclear neutrophil (PMN) counts in the endometrium, a total of 162 samples were collected from 54 estruses of 47 mares (before ovulation, day 0, and day 2) using a double-guarded cytology brush. Dinoprost (PG; 5 mg) was administered intramuscularly (i.m.) only once, on day 0, whereas oxytocin (OT; 20 U i.m.) was administered three times at 12-hr intervals starting on day 0. The plasma progesterone concentrations from days 0 to 14 were not different between the non-treatment (CON), PG treatment, and OT treatment groups. The PMN counts increased in all the groups from before administration to day 0 (CON, 0.90 to 3.55; PG, 1.20 to 8.45; and OT, 0.70 to 1.70; P=0.0014, 0.0046, and 0.0073, respectively). There was a significant decrease in PMNs from day 0 to day 2 only in the PG group (P=0.0073). The pregnancy rate was not different among the CON (12/18), PG (14/18), and OT (10/18) groups. The results of this study indicate that dinoprost can reduce uterine polymorphonuclear neutrophil counts.     

Blood and Intrauterine Leukocyte Profile and Function in Dairy Cows that Spontaneously Recovered from Postpartum Endometritis

The profile and function of blood and uterine leukocytes were evaluated in 14 dairy cows that spontaneously recovered from postpartum endometritis (mild, n=6 and heavy, n=8; general health not affected). From a minimum of 2 weeks before parturition until 6 weeks postpartum, blood samples were obtained twice weekly for leukocyte counts and leukogram determination and once weekly for flow cytometry assessment of polimorphonuclear neutrophils (PMN) phagocytic capacity and oxidative burst activity. Uterine fluid‐stained smears, obtained twice weekly from parturition until fluid was present in the uterus, were used for determination of the percentage of PMN, of phagocytizing PMN (phago‐PMN) and of the mean number of phagocyted bacteria per phagocytizing PMN (phagocytic index; PI). Uterine swabs were obtained twice weekly from parturition until 35 days postpartum for bacteriological examination. The time of endometritis diagnosis was similar in cows with mild or heavy endometritis but the latter cows had a significantly longer persistence of the infection and of the isolation of Gram‐negative anaerobes from the uterus. However, the effect of group (mild versus heavy) was not significant for all the blood and uterine parameters analysed. The effect of sampling day (within group effect) was significant (p<0.01 to p<0.00001) for all parameters, except for the blood monocyte count and the blood PMN phagocytic capacity, in which only a tendency for significance was observed (p<0.1). The effect of the interaction group × sampling day was significant only for the blood monocyte count. The phago‐PMN and the PI were significantly correlated (r=0.70, p<0.001). A significant correlation was also observed between the uterine fluid phago‐PMN and the blood PMN oxidative burst activity (r=?0.41, p<0.05). At the spontaneous recovery, the blood PMN oxidative burst activity was significantly higher (p<0.05) and the percentage of intrauterine phago‐PMN and the PI were significantly lower (p<0.001 and p<0.01, respectively) than at diagnosis of endometritis. These results suggest that a decrease in blood PMN oxidative burst activity until the first week postpartum could be associated with an increased susceptibility to early postpartum endometritis. The later increase in this parameter as well as the increase in the intrauterine fluid phago‐PMN and PI, might favour the spontaneous resolution of endometritis.     

PGD2/DP1途径对细菌感染奶牛子宫内膜组织中HMGB-1和PAFR表达的影响

为了研究前列腺素D₂（prostaglandin D₂,PGD₂）/DP₁受体途径对大肠杆菌（Escherichia coli）和金黄色葡萄球菌（Staphylococcus aureus）感染奶牛子宫内膜组织中炎症介质HMGB-1和PAFR的表达及对组织损伤程度的影响,试验以体外培养奶牛子宫内膜组织为研究对象,应用1×10^-6 mol/L DP₁受体激动剂（BW-245C和15d-PGJ2）和等量（1×10⁶ CFU/mL）大肠杆菌、金黄色葡萄球菌共同处理奶牛子宫内膜组织,采用实时荧光定量PCR、免疫组化和HE染色法检测奶牛子宫内膜组织中HMGB-1和PAFR的表达并评价组织损伤情况。结果显示,与空白对照组相比,大肠杆菌和金黄色葡萄球菌感染奶牛子宫内膜组织中HMGB-1和PAFR表达量显著升高（P<0.05）,而DP₁受体激动剂与大肠杆菌和金黄色葡萄球菌共处理奶牛子宫内膜组织中DP₁受体激动剂显著抑制奶牛子宫组织中HMGB-1和PAFR的表达（P<0.05）。HE染色结果显示,大肠杆菌与金黄色葡萄球菌感染的奶牛子宫内膜组织上皮细胞和腺上皮细胞完全脱落、坏死、崩解;而DP₁受体激动剂、大肠杆菌、金黄色葡萄球菌共同处理奶牛子宫内膜组织中,DP₁受体激动剂的加入显著减轻奶牛子宫内膜组织的损伤程度（P<0.05）。免疫组化染色法结果与以上两种方法结果一致。结果表明,PGD₂能够抑制大肠杆菌与金黄色葡萄球菌感染的奶牛子宫内膜组织中损伤相关因子HMGB-1、PAFR的表达,减轻组织损伤程度,这一作用可能是由DP₁受体所介导的。     

Resident Macrophages and Lymphocytes in the Canine Endometrium

Resident immune cells play a major role in endometrial immunity and in tissue homoeostasis. This study aimed to analyse the distribution of macrophages, B and T lymphocytes (respectively, Mø, B‐Lym and T‐Lym) in the canine endometrium throughout the oestrous cycle and in late involution (at the proestrus stage post‐parturition). An immunohistochemistry technique was used on samples from 50 post‐pubertal healthy female dogs, of which five in late post‐partum. The distribution of resident immune cells was analysed in three endometrial layers (superficial, intermediate and basal areas). Mø, B‐Lym and T‐Lym were demonstrated to reside in the endometrium in all the stages of the canine cycle; their numbers being considerably higher during late involution. T‐Lym were scattered in the stroma or amidst the glandular epithelium, constituting the predominant immune cell population in anestrus and proestrus, but decreased in number at all other stages. Endometrial B‐Lym remained fairly constant during the canine cycle, although its numbers were higher in late involution. Mø counts were higher during anestrus compared to the other stages, the cells being displaced into the superficial endometrial layer. Mø demonstrated the highest level in late involution samples, forming small aggregates below the surface epithelium. The number of immune cells was not normally distributed, suggesting the influence of individual factors, such as age or parity, not explored herein due to limited sample availability. Still, this study provides important information for the interpretation of endometrial biopsies in dogs and for the understanding of the increased susceptibility to uterine infection during dioestrus found in the bitch.     

Effects of induced endometritis on uterine blood flow in cows as evaluated by transrectal Doppler sonography

This study was conducted to evaluate the effects of induced endometritis on uterine blood flow in cows. Transrectal Doppler sonography was performed on uterine arteries of six cyclic cows before and for 4 days after inducing acute endometritis by intrauterine infusion of 720 mg of policresulen, and for 4 days of the following estrous cycle. Time-averaged maximum velocity (TAMV) increased (p < 0.001) and pulsatility index (PI) decreased (p < 0.0001) within 1 h of policresulen administration, and did not change (p > 0.05) in the next 4 days of the same cycle. TAMV and PI values in the subsequent cycle did not differ (p > 0.05) from the values measured before infusion and showed no changes (p > 0.05) within the cycle. Blood flow parameters were not related (p > 0.05) to plasma concentrations of progesterone and estrogen. All cows showed an acute endometritis determined by histopathological findings of biopsy samples taken 1 day after infusion and fibrotic endometrial alterations detected in the subsequent cycle. No relationships were observed between fibrotic changes of the endometrium and uterine blood flow during either cycle. In conclusion, acute inflammation is accompanied by a rise in uterine blood flow, but fibrotic alterations do not seem to be related to Doppler sonographic findings.     

Effects of Ethanol on Synthesis of Prostaglandin F2α in Bovine Females

Ethanol stimulates the production of prostaglandins in many species. The purpose of this study was to verify the effect of ethanol on the production of prostaglandin F2α (PGF2α) and luteolysis in bovine females. In the first experiment, Holstein cows at day 17 of the oestrous cycle were treated with 100% ethanol (0.05 ml/kg of body weight, IV; n = 5), saline (0.05 ml/kg of body weight, IV; n = 4) or synthetic prostaglandin (150 μg of D‐cloprostenol/cow, IM; n = 4). The plasma concentrations of 13, 14‐dihydro‐15‐keto PGF2α (PGFM; the main metabolite of PGF2α measured in the peripheral blood) were assessed by radioimmunoassay (RIA). There was an acute release of PGFM in response to ethanol comparing to other treatments (p ≤ 0.05). However, only cows treated with PGF2α underwent luteolysis. In the second experiment, endometrial explants of cross‐bred beef cows (n = 4) slaughtered at day 17 of the oestrous cycle were cultured for 4 h. During the last 3 h, the explants were cultured with medium supplemented with 0, 0.1, 1, 10 or 100 μl of 100% ethanol/ml. Medium samples were collected at hours 1 and 4 and concentrations of PGF2α were measured by RIA. Ethanol did not induce PGF2α production by the endometrium. In conclusion, ethanol does not cause luteolysis in cows because it stimulates production of PGF2α in extra‐endometrial tissues.     

Glycogen in Leukocytes from Bovine Blood and Milk

Glycogen content was determined quantitatively by the Anthrone reagent method in leukocytes obtained from blood and milk of five cows. Distribution of glycogen in leukocytes was studied by microscopic examination of slides stained by Periodic acid-Schiff (PAS) reaction. Blood glucose concentrations were investigated in these animals by standard procedures. In two of five cows both blood glucose levels and blood leukocyte glycogen levels on the same day were determined for six consecutive days. One hundred and two blood leukocyte samples from five cows had a mean glycogen content of 1.32 ± 0.04 (S.E.) mg/10⁹ WBC, and 6.11 ± 0.17 (S.E.) mg/10⁹ PMNs. Leukocyte preparations from 80 samples of milk comprising 97 to 98% PMNs contained 3.81 ± 0.18 (S.E.) mg glycogen/10⁹ milk leukocytes. In PAS preparations of blood and milk leukocytes glycogen was found almost exclusively in PMNs. Glycogen granules, present frequently in PMNs and occasionally in monocytes and large lymphocytes from blood, were not observed in those from milk. The glycogen level in milk leukocytes was significantly lower (P = <0.01) than that of the blood PMNs in every cow, and the overall mean difference between levels for milk leukocytes and blood PMNs was highly significant (P = <0.001). Mean blood glucose concentration in the five cows was 44.46 ± 0.66 (S.E.) mg%. There was no significant relationship between blood glucose and blood leukocyte glycogen levels in the five corresponding cows; nor between blood glucose and blood PMN glycogen levels on the same day in either of two cows investigated. Leukocyte preparations from milk samples obtained on the second day following intramammary infusion of endotoxin consistently contained markedly less glycogen than the leukocyte preparations from first day post-infusion samples.

These tended to level off and became intermediate between first and second day levels. It is postulated that the poor phagocytic competence of leukocytes from bovine mammary glands compared to their counterparts in blood observed by various workers may be due partially to low energy reserves in these cells.

     

Phagocytosis of bovine blood and milk polymorphonuclear leukocytes after ozone gas administration in vitro

To determine the effects of ozone on the phagocytosis of bovine polymorphonuclear leukocytes (PMNs), ozone gas was administered in vitro on the blood and milk of healthy lactating cows, cows with acute mastitis, and cows with milk fever. In the blood of healthy dairy cattle, although there was no significant effect of ozone gas on the viability of the leukocytes, phagocytosis of PMNs significantly decreased. In contrast, ozone gas administration in vitro significantly increased phagocytosis of PMNs from the blood of cows with acute mastitis and milk fever, and from mastitic milk. These findings showed that ozone administration in vitro has positive and negative effects on bovine PMN phagocytosis, depending on the health status of the animal.