Mastitis effects on reproductive performance in dairy cattle: a review

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1 Trop Anim Health Prod (2017) 49: DOI /s REVIEWS Mastitis effects on reproductive performance in dairy cattle: a review Narender Kumar 1 & A. Manimaran 1 & A. Kumaresan 2,3 & S. Jeyakumar 1 & L. Sreela 3,4 & P. Mooventhan 3,4 & M. Sivaram 1,5 Received: 27 July 2015 /Accepted: 22 February 2017 /Published online: 11 March 2017 # Springer Science+Business Media Dordrecht 2017 Abstract The reproductive performance of dairy animals is influenced by several factors, and accumulating lines of evidence indicate that mastitis is one of the determinants. Most of the published information relating mastitis with reproduction has evolved based on retrospective approach rather than controlled clinical studies. The complex nature of both mastitis and reproduction could be a limiting factor for understanding their relationship in detail. In this review, we analyzed the available retrospective studies on the effects of clinical mastitis on reproductive function and explained the possible mechanisms by which mastitis affects reproduction in dairy animals. Keywords Conception rate. Days open. Days to first service. Mastitis. Reproductive performance. Dairy animals Introduction Reproductive efficiency is one of the most important determinants of profitability in dairying. Over decades, reproductive efficiency in dairy cattle has declined across the world (Lucy 2001). Among several causes, infertility due to peri- and postpartum complications is a well-known factor for reduced reproductive efficiency. Recent lines of evidence indicate that the effects of mastitis are not restricted to udder but also extend to reproductive organs, thereby affecting the reproductive efficiency in dairy cattle. Clinical mastitis (CM) is a common and economically important disease in dairy animals because of reduced milk production, altered milk quality, increased involuntary culling rates, and posttreatment disposal of milk. The relationship between mastitis and reproduction is reported by several authors through retrospective studies (Barker et al. 1998; Gunay and Gunay 2008). Available information indicate that mastitis leads to decreased pregnancy rates, aberrations in the estrous cycle (Moore et al. 1991), early embryonic mortality or abortions (Risco et al. 1999), prolonged days open (Barker et al. 1998; Gunay and Gunay 2008), higher number of services per conception, and decreased conception rate (Kelton et al. 2001; Hertl et al. 2010) in dairy cattle. However, controlled clinical studies on understanding the * S. Jeyakumar jeyakumarsakthivel@gmail.com M. Sivaram sivaram.ndri@gmail.com Narender Kumar nklangyan@gmail.com A. Manimaran maranpharma@gmail.com A. Kumaresan ogkumaresan@rediffmail.com L. Sreela sreela312@gmail.com P. Mooventhan agriventhan@yahoo.co.in Livestock Research Centre, Southern Regional Station (SRS), ICAR-National Dairy Research Institute (NDRI), Adugodi, Bengaluru , Karnataka, India Theriogenology Lab, ICAR-NDRI, Karnal , India Livestock Research Centre, ICAR-National Dairy Research Institute (NDRI), Karnal , India ICAR-National Institute of Biotic Stress Management, Baronda, Raipur , Chhattisgarh, India Dairy Economics and Statistics Section, Southern Regional Station, ICAR-NDRI, Adugodi, Bangalore , India

2 664 Trop Anim Health Prod (2017) 49: pathogenesis of mastitis on reproduction are limited. Wilson et al. (2008) suggested that different types of mastitis pathogens (gram-negative and gram-positive bacteria) showed differential effects on the reproductive performance. The effect of time of mastitis occurrence (before or after AI), type and virulence of pathogens, and severity or duration of infection (acute severe or chronic subclinical, etc.) on reproduction still remains controversial. Recently, we observed that the occurrence of mastitis prior to and/or after first service resulted in extended days open in Zebu cattle and Murrah buffaloes (Manimaran et al. 2014). The present review consolidates the available information on the effects of mastitis on reproduction and attempts to explain the possible mechanisms by which mastitis influences reproductive performance in dairy animals. Additionally, other confounding factors determining the intensity of the ill effects of mastitis on reproduction are also discussed. Possible mechanisms by which mastitis affects reproduction Poor reproductive performance in mastitis-affected cows could be due to altered hormonal profile, oocyte competence, fertilization failure, and unfavorable uterine environment for embryonic development. Although the causes of early embryonic loss after fertilization or before implantation are multifactorial, few studies indicated that infectious disease or activation of immune responses elsewhere in the body (outside of the reproductive tract) might have an impact on the survival of early embryo. For instance, in response to the anecdotal statement of bovine practitioners about the relationship between CM and pregnancy rate, a preliminary study by Cullor (1990) revealed an altered interestrus interval (less than 18 days or more than 24 days) in CM cows; however, the effects varied with mastitis pathogen and age of the animal (Moore et al. 1991). It was suggested that endotoxin might induce luteolysis through the release of prostaglandin F 2 α (PGF 2 α) and other inflammatory mediators thus affecting early embryonic survival (Moore and Connor 1993). Mastitis and embryonic survival Hansen et al. (2004) suggested a comprehensive mechanism by which infection in mammary gland reduces the embryonic survival. They suggested an increased cytokine concentration as a key modulator of reproductive function through elevated body temperature (hyperthermia), disruption of the oocyte maturation and embryonic development, altered uterine function, and disruption of the hypothalamic-pituitary axis in dairy animals (Fig. 1). Elevated body temperature (pyrexia or fever) has been reported in both gram-positive- and gram-negative-induced mastitis (Wenz et al. 2001). The adverse effects of heat stress on reproductive function are a known phenomenon, and in fact, several in vitro (Edwards and Hansen 1997; Krininger et al. 2002) and in vivo (Al-Katanani et al. 2002; Gendelman et al. 2010) studies suggested that elevated body temperature may directly alter the developmental competence of oocyte and embryo or indirectly affect reproductive performance through decreased feed intake and body condition (Maltz et al. 1997). Role of cytokines on early embryo survival in mastitis-affected cows Nakajima et al. (1997) found higher levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in the serum and milk of cows affected with coliform mastitis and suggested that these cytokines might play an important role in inducing udder inflammation. Waller et al. (2003) explored the kinetics of neutrophils, cytokines (IL-8, TNF-α, IL-1β), and interferon-γ in milk and lymph of the supramammary lymph node after intramammary infusion of endotoxin and found that IL-8 plays a major role in the recruitment of neutrophils into the mammary gland. They suggested that IL-1β and TNF-α were not necessary for IL-8 production and release in response to endotoxin. Hoeben et al. (2000) studied differential kinetics, composition, and amounts of cytokines released into the mammary gland and in circulation of experimentally induced coliform mastitic cows and suggested that endotoxin might not be directly responsible for the systemic changes. Further, elevated concentrations of nitric oxide (NO) and PGF 2 α were also reported in milk of cows with experimentally induced Escherichia coli mastitis (Bouchard et al. 1999; Blum et al. 2000). Increased concentrations of these molecules in mastitic cows adversely affect the embryonic development. For instance, addition of TNF-α to bovine oocytes before or after fertilization increased the apoptosis and thus decreased embryonic development (Soto et al. 2003). This proapoptotic effect was suggested in a rat model also (Wuu et al. 1999). Similarly, the negative effects of TNF-α on embryonic development during in vitro or in vivo conditions were reported in cattle (Seals et al. 1998; Soto et al. 2003). Deb et al. (2011) reported that in vitro exposure of bovine oocytes to TNF-α reduced the number of oocytes developing to the blastocyst stage on day 8. Jackson et al. (2012) reported that detrimental effects of TNF-α on in vitro development of bovine embryos might be mediated through prostaglandin. However, the concentrations of TNF-α used in their experiment were higher than the reported serum levels in mastitic cows, and hence, further studies on prostaglandin concentrations in uterine or oviduct fluid of mastitic cows are important. Moreover, it is not clear whether mastitis is responsible for the elevation of PGF 2 α and NO synthesis in uterine tissue. On other hand, elevation of these molecules in the uterus of mastitisaffected animals cannot be ruled out, as the synthesis of these

3 Trop Anim Health Prod (2017) 49: Fig. 1 Flow diagram showing the possible mechanism on the effect of mastitis on reproduction in dairy cattle Mastitis Release of endotoxin/exotoxin UDDER Mastitis causing pathogens S. aureus E. coli etc. Systemic or localised release of inflammatory mediators Increased levels of cytokines like TNF ILS, PGS, and NO Direct effect on BRAIN Cortisol level Elevated body temperature (fever) OVARY Hypothalamo -Pituitary- Ovarian axis Altered GnRH, FSH, LH release & steroidogenesis Reduced reproductive behaviour / heat signs Altered follicular growth Poor oocyte maturation Decreased E2 synthesis Delayed or anovulation Poor oocyte quality Poor CL development Premature luteolysis Decreased P4 production UTERUS Uterine PGF2 release Reduced embryo / foetal growth Early Embryonic Mortality Fertilization failure Abortion Short cycle /Altered cyclicity Repeat breeding molecule is regulated by cytokines that were shown to be increased (TNF-α, nitrite, and nitrate) in the milk and plasma upon E. coli-induced mastitis (Blum et al. 2000) and thereby in the endometrium (Skarzynski et al. 2000). Accordingly, an increased PGF 2 α concentration in the endometrium could cause premature luteolysis. For example, induction of mastitis by Streptococcus uberis increased serum concentration of PGF2α metabolite (PGFM) in response to oxytocin treatment and intrauterine infusion of E. coli and caused luteal regression and shortened estrous cycle in heifers (Hockett et al. 2000; Gilbert et al. 1990). Giri et al. (1990) evaluated the effects of E. coli endotoxin infusions (1.0 or 2.5 μg/kg over 6 h) during the first, second, and third trimesters of gestation and found a dose-dependent increase in autacoids (PGs and thromboxane B 2 ) and cortisol concentrations. Hansen et al. (2004) also reported that intramammary infusion of lipopolysaccharides (LPS) and Str. uberis caused an increase in PGF 2 α, TNF-α, and NO concentrations in blood or milk and

4 666 Trop Anim Health Prod (2017) 49: premature luteal regression in cows. The detrimental effect of CM on early embryo development could be the result of elevated inflammatory mediators like cytokines leading to elevated levels of NO and PGF 2 α. Mastitis and altered endocrine milieu Altered fertility in mastitis-affected cows could be due to alterations in the secretion of hypothalamic-pituitary axis hormones responsible for oocyte maturation, ovulation, and luteal function. Administration of LPS near the estrus was shown to inhibit the luteinizing hormone (LH) surge leading to anovulation, delayed ovulation, and cyst formation in heifers. In support to this, a study (McCann et al. 1997) indicated that cytokines released following endotoxin challenge blocked the pulsatile secretion of LH or inhibition of gonadotropinreleasing hormone (GnRH) pulse amplitude. Consequently, insufficient follicular development and oocyte maturation could lead to insufficient estrogen production and thus lack of behavioral estrus, anovulation, and failure of conception. The role of cortisol in delayed or reduced LH surge was also suggested by many researchers (Li and Wagner 1983; Padmanabhan et al. 1983), and indeed, the concentration of this hormone is increased during mastitis (Hockett et al. 2000). Pathogen-specific mastitis effect on estrous cycle and follicular development Several authors reported that the gram-positive or gramnegative characteristics of CM pathogens did not differ in their effects on reproduction (Schrick et al. 2001; Santos et al. 2004). Similarly, Risco et al. (1999) reported higher early embryonic loss or abortion in mastitic cows, but no difference was found between types of pathogen causing CM. They suggested that gram-positive pathogens can also induce endotoxin-like effects on luteal function. However, Huszenicza et al. (1998) reported significantly higher rate of premature luteolysis in gram-negative pathogen-induced mastitis compared to gram-positive pathogens. Further, if the mastitis occurred during the follicular phase, the duration of cycle was lengthened in endotoxin mastitis compared to grampositive mastitis. The interestrus interval in mastitis-affected animals was also influenced by the causative organism; in mastitis due to Staphylococcus aureus, the interestrus interval was not altered, while in case of mastitis due to E. coli, the interestrus interval was altered. Kulcsár et al. (2004) studied the endocrine alterations in natural mastitis cases and found that in gram-negative mastitis, the endocrine changes were more obvious in the first 2 weeks after calving, while no significant changes were found in gram-positive mastitis. It could be inferred that in postpartum cows, mastitis due to gram-negative organisms may induce similar endocrine alterations as was observed in experimentally induced mastitis; however, in naturally occurring mastitis, these changes could vary within a wide range and may be more extended and robust. Lavon et al. (2010) found that naturally occurring chronic subclinical or acute clinical mastitis resulted in delayed ovulation associated with low plasma estradiol and a low or delayed preovulatory LH surge in 30% of cows affected with subclinical chronic mastitis. Consequently, they evaluated the function of preovulatory follicle in cows experiencing subclinical mastitis or a past event of acute CM and found abnormal steroidogenesis in one third of animals without any alteration of follicular growth dynamics. They also found no carryover effect of past CM on follicular function (Lavon et al. 2011a). Lavon et al. (2011b) evaluated the immediate and carryover effects of mastitis induced by gram-negative endotoxin (E. coli LPS) and gram-positive exosecretions (Sta. aureus ex.) on preovulatory follicle function. They found LPSinduced immediate, short-term, but not long-term impairment of follicular responses, while Sta. aureus-induced mastitis exhibited both immediate and carryover disruptive effects on preovulatory follicle function. Effect of mastitis on conception rate Hertl et al. (2010) found that regardless of the type of CM occurring 15 days prior or 36 days, post-ai did not affect conception rate. However, the occurrence of CM between 14 and 35 days post-ai resulted in a significant effect on probability of conception. CM due to gram-negative pathogen occurring between 8 and 14 days prior to AI was associated with a 32% reduction in conception rate, while CM due to grampositive and gram-negative pathogens from 1 to 7 days prior to AI was associated with a 50% reduction in probability of conception. Similarly, all types of CM occurring from 0 to 7 days post-ai were associated with reduced conception rates. Gram-positive CM reduced the probability of conception by about 47%, while gram-negative CM reduced up to 80%. Recently, Fuenzalida et al. (2015) reported that severity of mastitis was more important than etiology, but regardless of severity, microbiologically negative cases were not associated with reduced probability of pregnancy. Effect of mastitis on LH release and ovulation Several researchers studied the effect of immune or inflammatory stress induced by intravenous or intrauterine administration of LPS in cows (Peter et al. 1989; Suzukietal.2001) during the follicular phase and found suppressed pulsatile LH secretion and delayed or blocked preovulatory LH surge. The suggested mechanisms include depression of pituitary pulsatile LH release resulting in disruption of gonadotropin support

5 Trop Anim Health Prod (2017) 49: for follicular function; preovulatory estradiol secretion, which subsequently reduces the estrus expression; LH secretion; preovulatory LH surge; and ovulation. Since the follicular phase is quite a long duration (10 11 days), the actual effect of mastitis on ovulation may be different with proximity of mastitis and ovulation. Indeed, several studies showed that the exact time of exposure to the endotoxin prior to the LH surge is important for delayed ovulation (Battaglia et al. 2000; Breen et al. 2004). Lavon et al. (2008) studied the effects of intramammary or intravenous administration of LPS during estrus or at the time of ovulation on reproductive function. They reported that the LH surge was delayed in cows exposed to LPS during estrus resulting in delayed ovulation (delayed estrus-ovulation interval from 30 to 75 h), which ultimately reduced the chances of successful fertilization. They also found a large variation in LPS response among injected cows. A similar variability in disruption of LH pulse secretion and inhibition of LH surge was also reported in a small ruminant model (Breen et al. 2004; Battaglia et al. 1999). Variability in inflammatory response or impairment of reproductive functions after injection of Str. uberis was also reported in cattle (Hockett et al. 2000). Lavon et al. (2008) suggested that the level of milk yield, the body condition score, the stage of lactation, as well as the extent of increased milk somatic cell count (SCC), body temperature, heart rate, and respiratory rate as possible reasons for variation after LPS infusion. Asaf et al. (2013) found a differential disruptive effect of mastitis induced by gram-negative and gram-positive pathogens on oocyte developmental competence along with alterations in maternal gene expression. Furman et al. (2014) established an experimental model for subclinical mastitis induced by gram-positive (Sta. aureus origin) or gram-negative (endotoxin of E. coli) pathogens and found disruption of follicular functions, particularly in early antral follicles, due to subclinical mastitis. Effect of CM on oocyte developmental competence Roth et al. (2013) examined the effects of naturally occurring mastitis on in vitro oocyte developmental competence through their ability to undergo in vitro maturation, fertilization, and further development to the blastocyst stage in Holstein cows. They found that the mean percentage of embryos developed to the blastocyst stage on days 7 and 8 after fertilization was less in cows which had medium (311,000) and high (1,813,000) SCC (cells/ml milk) than in cows which had low (148,000) SCC. However, oocyte maturation did not differ between the bacterial types (gram negative or positive) involved or severity of infection (clinical or subclinical). Rahman et al. (2012) investigated the effects of naturally occurring chronic mastitis on ovarian function and found that severe CM cows had lower number of secondary follicles (>8 mm diameter) with decreased blood vessel density and higher fibrous stroma in the cortical area. However, the intensity of mastitis had no effect on the number of primordial (1 to 3 mm in diameter) and primary follicles (4 to 7 mm in diameter). They also found a lower level of growth and differentiation factor (GDF)-9 protein within the oocytes of different follicle sizes in severely affected cows. In contrast, Bromfield and Sheldon (2013) evaluated the effects of LPS on primordial ovarian follicle development and found that exposure of bovine ovarian cortex ex vivo to LPS reduced the primordial follicle pool. They also found that an acute exposure of mice to LPS reduced the primordial follicle pool along with increased follicle atresia, which was found to be TLR4 dependent. Wilson et al. (2008) investigated the reproductive performance following naturally occurring CM among J5 vaccinated cows. They found less pregnancy (38 42 vs 78%) and more days open (162 vs 131 days) in vaccinated cows compared to unvaccinated or control cows. Nguyen et al. (2011) suggested that cows with a high SCC (200, ,000 cells/ ml) had significantly higher incidence of prolonged luteal phase than cows with low SCC (50, ,000 cells/ml). Together, it may be inferred that despite variations in the nature of infection, types of pathogen, and severity of cases, mastitis had adverse effects on reproduction even in vaccinated cows. Therefore, understanding the pathophysiology of mastitis on follicular development and developmental competence of oocytes in naturally infected cows is very important to suggest a suitable therapeutic or preventive approach in these cows. Nonspecific effects of mastitis on reproduction The major problems in the evaluation of reproductive performance in mastitis-affected cows are difficulties in understanding all possible confounding factors responsible for the relationship between mastitis and fertility. For instance, poor reproductive performance of an individual animal (Nebel and McGilliard 1993) may be associated with either mastitis or higher milk yields (Windig et al. 2005). Therefore, studying the effects of mastitis on reproduction in cows with different capacities of milk production and under different management systems besides the prevailing agri-ecological conditions assumes significance. Further, advanced statistical models to handle these confounding factors are important to improve the statistical output and discuss about the problem. Recently, such techniques were used to understand the specific time of mastitis occurrence and its relation to reproductive performance (Perrin et al. 2007; Hertl et al. 2010), but the possibility of other diseases in CM-affected cows was not mentioned in these studies. However, Nava-Trujillo et al. (2010) reported that the extended days to first AI (about 53 days) were noticed when CM occurred at any time of the postpartum period in primiparous cows, while in multiparous cows, these adverse effects were observed only when CM

6 668 Trop Anim Health Prod (2017) 49: occurred over 62 days after calving, suggesting that the time of occurrence of CM and parity of the animal is very critical to understand the effects of mastitis on reproduction. Since experimentally induced mastitis by intramammary administration of either bacteria or endotoxin is mostly acute and short term in nature, the possible carryover effect of naturally occurring subclinical, chronic, and long-term mastitis on reproductive responses is not possible to study after experiment induction. Harman et al. (1996) reported lower probability of conception before 120 days in milk (DIM) in multiparous cows when they suffered acute but not chronic mastitis. Gómez-Cifuentes et al. (2014) assessed the association between CM, subclinical mastitis, body condition score, and reproductive performance of cows under pasture-based management system. They found that subclinical mastitis significantly increased the number of services, while CM did not affect reproductive performance in terms of the number of services or pregnancy rate suggesting that the nature of infection and its severity are important determinants of mastitis outcome. Since the relationship between mastitis and reproduction was initially suggested through retrospective studies (Barker et al. 1998; Santos et al. 2004), the possibility of other diseases in these CM cows cannot be ruled out. Fourichon et al. (2000) reported through meta-analysis of published studies that retained placenta (RP), ovarian cysts (OC), and metabolic disorders negatively affected reproduction, but mastitis had no effect on reproductive performance. Several other authors (Loeffler et al. 1999; Maizon et al. 2004) also reported that, other than mastitis, dystocia, RP, displaced abomasum, ketosis, milk fever (MF), metritis (ME), and pyometra negatively influenced the reproductive performance of dairy cows. Vacek et al. (2007) evaluated the relationship among several health disorders (MF, RP, ME, endometritis and pyometra, OC, and lameness) including CM and reproductive performance in dairy cows. They found that RP, OC, and ME had a significant effect on the days to first AI, days open, and service per conception (SC), while MF delayed days to first AI and lameness increased days open and SC. However, the relationship between CM and fertility parameters was not as explicit as the authors found that CM only increased days open and days to first service, without altering other fertility parameters. Ribeiro et al. (2013) reported that clinical and subclinical periparturient diseases showed an additive negative effect on reproduction. However, individually, mastitis did not alter the resumption of estrous cyclicity and pregnancy per AI on day 30 and day 65 after insemination in these cows. In contrast, Peake et al. (2011) reported that the combined incidence of lameness, subclinical mastitis, and body condition score loss causes delayed onset of first luteal phase from calving and had synergistic detrimental effects on progesterone profile in Holstein-Friesian cows. Morris et al. (2009) also found reduced fertility in dairy cows that suffered with these three production stressors. Ahmadzadeh et al. (2009) suggested that the effect of mastitis and other diseases is additive in nature, and thus, reproduction was affected to a greater extent when cows suffered both mastitis and other diseases than an independent event of diseases. Vacek et al. (2007) evaluated the influence of repeated episodes of CM and found that cows that experienced two or more incidence of CM had more days open and number of AI compared to healthy cows, while cows that suffered CM once did not differ significantly with those that had two or more incidences of CM during lactation. Moussavi et al. (2012) reported that increased number of mastitis episodes in early lactation significantly increased the service per conception with no apparent impact on the days open in heifers. A genetic role was also suggested by Heringstad et al. (2006)as they found that mastitis was genetically associated with reduced fertility, with a genetic correlation ranging between 0.21 and In contrast, few studies showed that CM and nonreturn rate within 56 days after first AI during first lactation were independent traits in Norwegian Red cows (Heringstad et al. 2006, 2009). Other possible variables such as parity, breeding season, and DIM as well as mastitis and breeding, type of breeding, etc. were also evaluated by some workers (Risco et al. 1999; Chebeletal.2004). Critical period in which mastitis adversely affects reproduction The period of occurrence of mastitis had been shown to influence the intensity of adverse effects on postpartum reproduction in cows. Barker et al. (1998) and Schrick et al. (2001) investigated the effect CM during different times of early lactation (CM before first AI and between first AI and pregnancy confirmation) and found that CM before AI increased the number of days to first AI, while CM after first AI increased days open (DO) and service index (number of AI required per conception). The results obtained by several workers on this aspect are summarized in Table 1. Collectively, it was suggested that CM during early lactation had a negative impact on the reproductive performance of dairy cows. In contrast, Gómez-Cifuentes et al. (2014) found no association between time of occurrence of CM and pregnancy rate or number of services per conception. Boujenane et al. (2015)reportedthat when CM was considered as fixed effect, it had significant effects on days to first service, while nonsignificant effects were observed on DO and service index. However, when it was classified based on the time of first CM occurrence (<60, 60 90, and >90 DIM), there was no significant effect on reproductive performance. Podpečan et al. (2013) reported that mastitic cows in the first 3 months postpartum did not differ significantly from the clinically healthy cows in terms of days to first service, first service to conception interval, and DO.

7 Trop Anim Health Prod (2017) 49: Table 1 The mastitis effects on reproductive performance in dairy cows References Reproductive parameters CM before first AI CM between the first AI and pregnancy confirmation Uninfected/control Barker et al. (1998) a Days to first AI 93.6 ± 5.6a NA 71 ± 2.2b Days open (days) ± 10.8a ± 13.3c 92.1 ± 4.6b Services per (no) conception 1.6 ± 0.3a 2.9 ± 0.3b 1.7 ± 0.1a Schrick et al. (2001) b Calving to first service 75.7 ± 1.8a 75.2 ± 4.4ab 67.8 ± 2.2b Days open ± 4.8a ± 11.4b 85.4 ± 5.8c Services per conception 2.0 ± 0.1a 3.1 ± 0.3b 1.6 ± 0.2c Santos et al. (2004) Days to first AI 68a 58b 64ab Days open 165 ± 5.7a 189 ± 7.2c 140 ± 3.7b Services per conception 2.6 ± 0.1a 3.0 ± 0.2b 2.6 ± 0.1a Conception rate at first AI 22%a 10%b 29%c Gunay and Gunay (2008) Calving to first service 95.2 ± ± ± 6.3 Days open ± ± ± 10.3 Services per conception 2.1 ± ± ± 0.8 Nava-Trujillo et al. (2010) c Days to first AI ± 5.22 NA ± 4.52 Days open ± ± 7.45 Services per conception 2.35 ± ± 0.16 Conception rate at first AI 49.72% 56.10% Yang et al. (2012) Days to first AI ± 1.23a ± 1.69b ± 0.34c Days open ± ± ± 2.17 Services per conception 1.88 ± 0.08a 2.19 ± 0.16b 1.53 ± 0.03c Conception rate at first AI 38.1%a 27.8%a 54.9%b Value with different lowercase letters within row differ at p<0.05 a The control group also includes CM-affected cows after pregnancy confirmation b Both clinical and subclinical mastitic cows c CM before first service They suggested that rapid response to treatment, better management, and inclusion of a smaller number of mastitic cows were possible reasons for the nonsignificant effects. An adverse effect of mastitis on reproduction was also studied through early embryonic mortality, pregnancy rate, and conception rate (CR) retrospectively in cows by several researchers (Table 1). Chebel et al. (2004) reported that the occurrence of CM between first AI and pregnancy confirmation was associated with 2.8 times more pregnancy loss. Higher incidences of abortions in mastitis-affected cows were also reported by other workers (Risco et al. 1999; Santos et al. 2004). Moore et al. (2005) reported that cows with higher linear SCC score (>4.5) before AI had higher embryonic loss by 35 to 41 days as compared to cows with a score <4.5. Lomander et al. (2013) indicated that cows with an increasing SCC after calving had a lower probability of pregnancy at first AI and had a higher number of inseminations per animal than healthy cows. Hockett et al. (2005) reported that cows with CM during preovulatory period had decreased expression of estrus, period of estrus, and pregnancy rate. Further, to understand its mechanism, they studied the effects of experimentally induced mastitis using Str. uberis before ovulation on endocrine profile and ovarian structure and found that estradiol remained at basal levels in cows following experimentally induced CM along with reduced LH pulsatality and LH surge, without affecting the follicle size. It ultimately decreased the estrous behavior and delayed establishment of pregnancy for approximately one estrus cycle. On other hand, the occurrence of mastitis after ovulation had minimal effects on reproductive performance. In contrast, when mastitis was induced during the luteal phase of early lactation ( 30 DIM), the cows (3 4 days after induction) had more concentration of cortisol on the 4th and 7th days, while the peak concentration of PGFM (after oxytocin challenge) was also increased in mastitic than control cows. However, no differences were found in the concentration of prolactin, LH (after GnRH challenge), and estradiol between mastitis and control cows (Hockett et al. 2000). These contrasting results clearly suggest the role of physiological status of the animals and the importance of proper experimental design to understand the effects of mastitis on reproduction. Huszenicza et al. (1998) studied the resumption of cyclicity in mastitis-affected and healthy cows between days 1 to 14 and 15 to 28 days postpartum. They found that cows that

8 670 Trop Anim Health Prod (2017) 49: suffered from any type of mastitis-causing organism (gram +ve or gram ve)during15to28dayspostpartumshowed delayed onset of ovarian cyclicity and estrus compared to cows that suffered mastitis during 1 to 14 days postpartum. It suggested that mastitis can affect the resumption of ovarian activity in postpartum cows when it occurs at the expected time of first ovulation, i.e., between 15 and 28 days postpartum. On the contrary, P 4 profiles during early lactation, pregnancy rate, and DO were not altered in these mastitic cows. Therefore, detailed studies on the effects of mastitis on postpartum cyclicity during early postpartum or breeding period are warranted. Perrin et al. (2007) found decreased CR when CM occurred 0 to 3 weeks before first AI, while CR was not affected when CM was observed 3 to 6 weeks before, 0 to 3 weeks after, or 3 to 6 weeks after AI, suggesting that CM primarily affects ovulatory factors and oocytes rather than products of conception (i.e., embryos). In contrast, Hertl et al. (2010) found that the occurrence of CM (especially gram-negative CM) immediately after AI was associated with very low probability of conception. It suggested that CM could also be interfering with oocyte fertilization or embryonic development. Hudson et al. (2012) found a negative association between CM and reproductive performance over a wide time frame relative to the risk period (from 28 days before to 70 days after the risk period) and reported that subclinical mastitis (individual cow SCC of >399,000/ml) was associated with decreased reproductive performance 30 days following a risk period or service. Similarly, Soto et al. (2003) studied the effect of LPS on oocytes before and after fertilization and found that LPS had deleterious consequences on both oocyte function and embryonic development. Klaas et al. (2004) reportedthat mastitis before AI had no effect on CR or DO, while Miller et al. (2001) reported that high SCC before AI had some effect on the nonreturn rate, which does not warrant the postponing of first service. Hockett et al. (2000) did not find any suppression of E 2 or LH when they induced mastitis during the luteal phase. Maizon et al. (2004) reported CM as one of the most important factors for extended DO when CM was diagnosed after 45 DIM. Pinedo et al. (2009) evaluated the effect of high ( 4.5) linear SCC score during early lactation on reproductive performance and found that the days to first AI were delayed by 21.8 in cows with at least one high linear SCC before the first AI, while cows with at least one high linear SCC before the fertile breeding had 48.7 more days to conception and 0.49 more services to conceive. They also found that the probability of pregnancy decreased by 44% and the risk of abortion increased (1.22) during the first 90 days of gestation if a high linear SCC occurred before breeding. Lavon et al. (2011c) evaluated the association of CR with the pattern and level of SCC elevation relative to time of insemination and found significantly reduced CR even in mild SCC elevation before AI. Recently, Filho et al. (2012) reported that those cows diagnosed as affected with CM up to 60 days postpartum had significantly the shortest calving to conception intervals (132.4 ± 7.2 days), followed by those diagnosed during days postpartum (153.9 ± 8.0 days) and by those diagnosed after 120 days postpartum (231.3 ± 9.9 days). It is possible that the cows diagnosed up to 60 days postpartum probably recovered with no negative effects for their subsequent reproductive performance. Conclusion Taken together, the existing information indicates that mastitis negatively affects the reproductive performance in cows. The adverse effects of mastitis on reproduction are mostly due to increased days to first insemination, days open, and decreased overall CR. Mastitis is also associated with early embryonic losses, abortion, and culling of animals. Advanced analytical methods revealed that mastitis causes more adverse effects during some critical period during postpartum. However, further studies in natural mastitis-affected animals are required to understand the pathophysiology of mastitis on reproductive function. Understanding the other confounding factors associated with mastitis is also equally important for the development of alternative breeding programs for cows when mastitis occurs during a critical period. Since antibiotic treatment is an unavoidable tool for mastitis management, an evaluation of its efficacy through fertility and other specific inflammatory markers would also increase the current understanding of reproductive performance in mastitis-affected animals. Acknowledgments The authors are thankful to the Director, ICAR- NDRI for providing the necessary facilities. Compliance with ethical standards Conflict of interest interest. References The authors declare that they have no conflict of Ahmadzadeh, A., Frago, F., Shafii, B., Dalton, J.C., Price, W. J. and McGuire, M. 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