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The establishment of the intestinal microbiota is critical for the digestive and immune systems. We studied the early development of the rectal microbiota in horse, a hindgut fermenter, from birth until 7 days of age, by qPCR and 16S rRNA gene amplicon sequencing. To evaluate initial sources of the foal microbiota, we characterised dam fecal, vaginal and oral microbiotas. We utilised an amplicon sequence variant (ASV) pipeline to maximise resolution and reproducibility. Stringent ASV filtering based on prevalence and abundance in samples and controls purged contaminants while preserving intestinal taxa. Sampled within 20 minutes after birth, rectal meconium contained small amounts of diverse bacterial DNA, with a profile closer to mare feces than mouth. 24 hours after birth, rectum was colonised by Firmicutes and Proteobacteria, some foals dominated by single genera. At day 7, the rectal genera were still different from adult feces. The mare vaginal microbiota contributed to 24 h and 7 day microbiotas. It contained few lactobacilli, with Corynebacterium, Porphyromonas, Campylobacter and Helcococcus as the most abundant genera. In the oral mucosa, Gemella was extremely abundant. Our observations indicate that bacteria or bacterial components are present in the intestine immediately after birth, but the newborn microbiota changes rapidly.

Matrix metalloproteinase (MMP)-2 and MMP-9 are gelatinases that take part in several reproductive processes. The aim of this study was to measure levels of MMP-2 and MMP-9 in fractionated stallion ejaculates, and to evaluate the association between these components and semen quality, and sperm longevity during cooled storage. Semen quality were assessed separately for sperm-rich fractions (HIGH), sperm-poor fractions (LOW), and whole ejaculate samples (WE) from 33 stallions. After cooled storage with SP either present or removed, sperm motility and DFI were determined. The relative activity of the pro-form of MMP-2, active MMP-2 and total MMP-9 were evaluated using gelatin zymography, and all were present in all fractions of the stallion's ejaculate, with higher relative activity of the latent than active forms and the highest relative activity in the HIGH fraction. The relative activities of MMP-2 and MMP-9 were positively correlated to sperm concentration and total sperm count, but only in the HIGH fraction and not in LOW or WE. The relative activities of MMPs were not related to differences in sperm longevity during cooled storage, measured as sperm motility and DFI. There was a harmful effect of SP on DFI during storage, but this effect was not associated with differences in the relative activities of MMPs. In conclusion, the relative activities of MMPs are not useful as markers for semen quality (other than sperm concentration), or sperm survival during storage in horses. (C) 2021 Elsevier Inc. All rights reserved.

Postpartum metritis in mares is a life-threatening condition associated with severe clinical signs due to endotoxemia, and it is often followed by complications such as laminitis. Repeated large-volume uterine lavages are commonly recommended as a part of the treatment protocol to remove endotoxin-laden contents from the uterus. It has, however, also been suggested that lavages may increase the uptake of endotoxin into the circulation, leading to a deterioration of clinical signs. Endotoxemia is associated with the release of a multitude of inflammatory mediators regulating the immune response. The aim of this study was to evaluate if uterine lavage influences serum levels of the inflammation markers soluble CD14 (sCD14), chemokine (C-C motif) ligand 2 (CCL2), and interleukin (IL)-10 in mares with postpartum metritis. Serum samples were collected from eight mares treated for metritis at a university teaching hospital. Mares with fever, tachycardia, and/or leukopenia and hemosanguineous or purulent intrauterine fluid within 1 week of foaling were included in the study. Serum samples were taken before uterine lavage and 15 and 30 minutes after starting the lavage. The concentrations of sCD14, CCL2, and IL 10 were determined with a fluorescent beadebased immunoassay. There were no significant differences between sCD14, CCL2, or IL-10 levels at different sampling times. Heart rate was significantly lower after uterine lavage than before. The differences in body temperature and leukocyte count before and after lavage were not significant. In conclusion, uterine lavage had no immediate effect on the serum concentration of sCD14, CCL2, or IL-10, nor did it affect clinical parameters negatively. (c) 2020 Elsevier Inc. All rights reserved.

Artificial insemination (AI) with cooled stallion semen has increased markedly during the last decades in all countries, but fertility is often lower than when fresh semen or natural mating is used. The objective of this study was to examine field data (1634,cycles 523 Standardbred (SB) mares, 575 Finnhorse (FH) mares, and 90 stallions) using multivariable logistic regression for factors influencing the pregnancy rate (PR) after Al with cooled transported semen from SB and FH stallions. The PR per cycle for the material was 47%: Finnhorses 42% and Standardbreds 53%. When assessed with multivariable logistic regression analyses with a generalized linear mixed model, variables that affected the PR were breed, the number of inseminated estrus cycles, the percentage of progressively motile sperm (PMOT) in the ejaculate/AI dose at the time of shipment, and the number of progressively motile sperm in the Al dose at the time of insemination. In Standardbreds, variables that increased the per cycle PR were the number of Al per estrus cycle (multiple inseminations increasing the probability of pregnancy compared to only one insemination), the number of inseminated cycles, and PMOT in the AI dose at the time of insemination. In Finnhorses, the number of Al per estrus cycle (two and three inseminations increasing the probability of pregnancy compared to only one), the number of spermatozoa in the ejaculate and in the Al dose, and PMOT in the ejaculate/AI dose at the time of shipment increased the per cycle PR. Non-significant factors for the whole material included the type of artificial vagina (open-ended or closed), transport time, place of Al (stud farm or home stable), insemination done by veterinarian or technician, weekday, month, age of the mare (all age classes combined), age of the stallion, ejaculate parameters (sperm concentration, total number of sperm), and insemination dose parameters (volume proportion of seminal plasma, sperm concentration, PMOT, total number of sperm). In conclusion, breed, breeding opportunity in more than one cycle, more than one insemination/estrus, PMOT of the ejaculate/AI dose and the number of progressively motile sperm in the Al dose at the time of insemination are important for the outcome of inseminations with cooled semen. (C) 2018 Elsevier Inc. All rights reserved.

Semen jets in the stallion's ejaculate differ in both the biochemical composition of seminal plasma (SP) and in sperm concentration, forming sperm-rich fractions (HIGH) and sperm-poor fractions (LOW). This study examined (i) the association of the levels of selected components of SP [alkaline phosphatase (AP), acid phosphatase (ACP), Na(+), Cl(-), K(+), Ca, Mg and prostaglandin E(2)] with semen quality, sperm longevity and fertility; (ii) the effects of the presence of SP on sperm survival during cooled storage; (iii) the differences in the composition of SP between sperm-rich and sperm-poor ejaculatory fractions; (iv) the differences between these fractions in the effects of SP on sperm longevity. The levels of AP, ACP and Na(+) were higher in HIGH compared with LOW, whereas higher concentrations of Ca, K(+) and Mg were found in LOW than in HIGH. None of the measured components were correlated to the first cycle pregnancy rate. The presence of SP during cooled storage caused DNA degradation, but total and progressive sperm motility was not affected. Sperm-rich fractions stored with low levels of SP maintained higher level of DNA integrity compared with sperm-rich fractions in higher levels of SP, or sperm-poor fractions irrespective of the proportion of SP. The concentration of K(+) in LOW may give some indication on the maintenance of total sperm motility during cooled storage.

Seminal plasma (SP) is the fluid portion of semen, secreted by the epididymides and the accessory glands before and during ejaculation. The stallion s ejaculate is a series of jets that differ in sperm concentration, semen volume and biochemical composition. Before the actual ejaculation, a clear and watery pre-sperm fluid is secreted. The first three jets form the sperm-rich fractions, and contain ¾ of the total number of sperm. The semen volume and sperm concentration in each of the jets decrease towards the end of the ejaculation, and the last jets are sperm-poor fractions with a low sperm concentration. The aims of these studies were to examine the effects of the different SP fractions, and the presence of SP, on sperm survival during storage. Pre-sperm fluid, and semen fractions with a high (sperm-rich) and low (sperm-poor) sperm concentration were collected in five experiments. The levels of selected enzymes, electrolytes and proteins in different SP fractions were determined. These studies also aimed at assessing the individual variation in the levels of the selected SP components and in the effects of SP on spermatozoa. The association between the components of SP and semen quality, sperm longevity, and fertility was examined with a stepwise linear regression analysis. Compared to samples containing SP during storage, centrifugation and the subsequent removal of SP reduced sperm motility parameters during 24 h of cooled storage in all SP fractions, but sperm membrane integrity was not affected. Some of the measured post-thaw motility parameters were also higher in samples containing SP compared to samples stored without SP. In contrast, the proportion of DNA-damaged spermatozoa was greater in the samples stored with SP than those without SP, and this effect was seen in both sperm-rich and sperm-poor fractions. There were no differences in DNA integrity between fractions stored with SP, but the sperm-rich fraction showed less DNA damage than the sperm-poor fraction after SP removal. The differences between fractions in sperm motility after cooled storage were non-significant. The levels of alkaline phosphatase, acid phosphatase and β-glucuronidase were higher in the sperm-rich fractions compared to the sperm-poor fractions, while the concentrations of calcium and magnesium were higher in sperm-poor fractions than in sperm-rich fractions. The concentrations of sodium and chloride were highest in pre-sperm fluid. In the sperm-poor fraction, the level of potassium was associated with the maintenance of sperm motility during storage. The levels of alkaline and acid phosphatase were associated with sperm concentration and the total number of spermatozoa in the ejaculates. None of the measured SP components were correlated to the first cycle pregnancy rate. In summary, the removal of SP improved DNA integrity after cooled storage compared with samples containing SP. There were no differences in the maintenance of sperm motility between the sperm-rich and sperm-poor fractions and whole ejaculates during cooled storage, irrespective of the presence of SP. The lowest rate of DNA damage was found in the sperm-rich fractions stored without SP. In practice, the results presented in this thesis support the use of individual modifications of semen processing techniques for cooled transported semen from subfertile stallions. Jäähdytettynä säilytetyn siirtosperman käyttö on viime vuosikymmenen aikana yleistynyt. Siittiöiden säilytyskestävyydessä on kuitenkin vaihtelua, eikä tähän vaikuttavia tekijöitä tunneta kovin hyvin. ELL Maria Kareskoski tarkastelee väitöstutkimuksessaan oriin seminaaliplasman vaikutuksia siittiöiden säilytyskestävyyteen, sekä seminaaliplasman koostumusta. Kareskoski osoittaa tutkimuksessaan, että seminaaliplasman osuuden pienentäminen ennen säilytystä vähentää säilytyksen aikaisia DNA-vaurioita siittiöissä. Oriiden välisen vaihtelun takia yksilölliset muutokset siemennesteen käsittelyssä ennen säilytystä voivat olla hyödyksi jos oriin siittiöiden säilytyskestävyys on heikkoa. Seminaaliplasma on siemennesteen nestemäinen osa, joka koostuu lisäsukurauhasten tuottamasta nesteestä. Sen ainesosat vaikuttavat moniin hedelmöitystä edeltäviin tapahtumiin. Oriin siemenneste koostuu useista osista, eli fraktioista. Ejakulaation alkuvaiheen siittiörikkaat fraktiot sisältävät suurimman osan siittiöistä, ja loppuvaiheen siittiököyhät fraktiot sisältävät vähemmän siittiöitä ja enemmän seminaaliplasmaa. Seminaaliplasman biokemiallinen koostumus vaihtelee näiden fraktioiden välillä, sillä eri lisäsukurauhasten tuottamaa nestettä vapautuu fraktioihin tietyssä järjestyksessä. Seminaaliplasman tilavuusosuuden vaikutus siittiöiden säilytyskestävyyteen Säilytyksen aikana seminaaliplasman läsnäolo oli yhteydessä siittiöiden DNA-vaurioihin. Siittiöiden DNA-vaurioita nähtiin vähemmän niissä näytteissä, joista suurin osa seminaaliplasmasta oli poistettu ennen säilytystä, verrattuna näytteisiin joissa noin kolmasosa tilavuudesta oli seminaaliplasmaa ja loput laimennusnestettä. Siittiöiden liikkuvuudessa säilytyksen jälkeen nähtiin paljon orikohtaista vaihtelua. Fraktioerot siittiöiden säilytyskestävyydessä ja seminaaliplasman koostumuksessa DNA-vaurioiden määrä lisääntyi säilytyksen aikana vähiten siittiörikkaassa fraktiossa josta suurin osa seminaaliplasmasta oli poistettu. Kun seminaaliplasmaa oli mukana näytteessä säilytyksen aikana, fraktioeroja siittiöiden liikkuvuudessa ja DNA-vaurioiden määrässä ei nähty. Tutkittujen entsyymien (alkalinen ja hapan fosfataasi, β-glukuronidaasi) pitoisuudet olivat korkeampia siittiörikkaissa kuin siittiököyhissä fraktioissa, kun taas kalsium- ja magnesiumpitoisuudet olivat korkeampia siittiököyhissä kuin siittiörikkaissa fraktioissa. Natriumia ja kaliumia oli eniten ennen ejakulaatiota erittyvässä esisekreetissä, joka ei sisällä siittiöitä. Siittiököyhän fraktion kaliumpitoisuudella saattaa olla merkitystä siittiöiden liikkuvuuden kannalta, mutta yksikään tutkituista ainesosista ei ollut yhteydessä oriin hedelmällisyyteen.

The most probable reason for persistent postbreeding endometritis in mares is weak myometrial contractility. The influence of oxytocin (OT; an ecbolic agent) and flunixin meglumine (FLU; a prostaglandin inhibitor serving as a model for mares with decreased uterine contractility) on uterine response to artificial insemination (AI) was studied in mares with no history of reproductive failure. The mares were treated intravenously with 10 mL saline (Group C, n = 10) or 0.01 IU/kg OT (Group OT, n = 10) 2, 4, 8, and 25 h after AI. Group FLU (n = 11) was treated with 1.1 mg/kg FLU 2 h after AI and with saline thereafter. The mares received the same treatments in the first and third cycles but were sampled either at 8 or 25 h. The amount of intrauterine fluid (IUF) and edema and the number of uterine contractions were recorded before AI and 10 min after the treatments using transrectal ultrasonography. At 8 h after AI, the mares were treated with human chorionic gonadotropin, and, after 8-h or 25-h scans, a 500-mL uterine lavage and a biopsy were performed. Ovulation was confirmed at 48 h and pregnancy 14 to 17 d after AI. No manipulations were done during the second estrus. At 8 h after AI, Group FLU had more polymorphonuclear leukocytes (PMNs) in the uterine lavage fluid than did Group OT (P < 0.05), but uterine contractions did not differ significantly. At 25 h, the PMN concentrations were low in all groups. Group OT rarely showed IUF. The uterine biopsy specimens of Group FLU showed less inflammation of the stroma but more PMNs in the uterine lumen 8 h after AI than that of the control group (P < 0.05). The pregnancy rates did not differ between the groups (63% C, 53% OT, and 50% FLU). Oxytocin rapidly and effectively removed IUF and PMNs after AI and thereby shortened the duration of postbreeding inflammation.

Seminal plasma is a mixture of secretions produced in the testes, epididymides and accessory sex glands, and ejaculated as several consecutive fluid fractions. The composition of seminal plasma and the effects on sperm longevity vary between fractions and individual stallions. This review focuses on the sequence of ejaculation, constituents of seminal plasma and their potential use as fertility markers as well as the influence of seminal plasma on spermatozoa during storage.