J. Dairy Sci. 105:2587–2596 https://doi.org/10.3168/jds.2021-21270 © 2022, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Responses to melatonin of 2 breeds of dairy ewes in early lactation under autumn photoperiod conditions A. Elhadi, A. A. K. Salama, X. Such, and G. Caja* Group of Research in Ruminants (G2R), Department of Animal and Food Sciences, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain ABSTRACT respectively). No differences in basal plasmatic melato- nin were detected between breeds (6.4 ± 1.1 pg/mL, on A total of 72 dairy ewes of 2 breeds (MN, Manchega, average), indicating the greater responsiveness to the 72.4 ± 1.9 kg of body weight, n = 36; LC, Lacaune, implants of the lighter MN ewes. Plasmatic prolactin 77.7 ± 2.3 kg of body weight; n = 36) were used to tended to decrease in the MEL-treated ewes (−35%, on evaluate the lactational effects of melatonin implants average), but the effect was significant only in the MN in early lactation and under the short-day photoperiod ewes (−54%), in agreement with their greater response conditions of autumn (experiment was centered on to MEL. No effects of MEL treatment were detected the winter solstice). Ewes lambed in autumn and were on plasmatic IGF-I in either breed. Moreover, body penned indoors in 12 balanced groups of 6 ewes by reserves did not vary by effect of MEL treatment or breed, body weight, age, and number of lambs, and breed throughout the experiment. In conclusion, the randomly assigned to a 2 × 2 × 3 factorial design use of exogenous melatonin as MEL implants, together (treatment × breed × replicate). Ewes suckled their with the endogenous melatonin naturally produced un- lambs for 28 d. Treatments were (1) melatonin (MEL), der short-day photoperiod conditions, had no effects on which received 1 subcutaneous implant of melatonin the early-lactation performances of dairy sheep, despite (18 mg/ewe) in the ear base at 35 ± 1 d (1 wk after their breed and level of production. lamb weaning), and (2) control, which did not receive Key words: dairy sheep, early lactation, melatonin, any treatment. Ewes were fed ad libitum a total mixed prolactin ration (forage:​concentrate, 60:40) and machine milked twice daily. Daily milk yield was automatically re- INTRODUCTION corded from d 29 to 105 of lactation and sampled every 2 wk for composition. Jugular blood was sampled for Sheep are seasonal breeder animals conditioned plasma hormone analyses at 30, 50, 80, 110, and 124 by photoperiodic signals that use melatonin (MEL) d of lactation. Body reserves were assessed every 2 wk. as a neuroendocrine transmitter for informing on the Feed intake was measured by pen during 3 separated light:​dark pattern (Bittman et al., 1983; Borjigin et periods after the start of the treatments (wk 2 to 3, wk al., 1999; Reiter et al., 2009). Melatonin is present in 6 to 7, and wk 10 to 11). Feed intake, and milk yield near all animal lineages and mainly secreted during the and composition varied by breed, but no MEL effects night into the cerebrospinal fluid (central) by the pineal were detected on dry matter intake, milk yield, milk gland and the visual system (retina, Harderian gland; composition, or fat and protein standardized milk in ei- Reiter, 1980, 1991). Extrapineal secretion (peripheral) ther breed. As a result of the unique composition of the and storage of MEL in endocrine (e.g., gut, liver, kid- implants and the variable body weights of the ewes, the ney, and so on) and nonendocrine (e.g., thymus, mast MEL treatment dose (on average, 0.24 mg/kg of body cells, leucocytes, and so on) cells has also been proved weight) was 6.8% greater in the MN (lighter) than in (Kvetnoy, 1999), but is unlikely to be dependent on the LC (heavier) ewes. Plasmatic melatonin markedly light. increased in the MEL-treated ewes (on average, 111%), Light-dark treatments and administration of exog- but despite the amount of MEL used, the MN respond- enous MEL to advance the breeding season and to ed greatly compared with the LC ewes (150 vs. 63%, improve the reproductive performances of small rumi- nants have been largely investigated. Administration of MEL has been shown to advance the beginning of Received September 10, 2021. Accepted November 20, 2021. the natural breeding season from the end of summer to *Corresponding author: gerardo.caja@​uab​.es spring by mimicking the stimulatory effect of autumnal 2587 Elhadi et al.: MELATONIN IN DAIRY EWES 2588 short days in sheep and goats (Haresign et al., 1990; and, on average, greater in the long-day than in the Williams et al., 1992; Zarazaga et al., 1998). Moreover, short-day MEL-treated lactating ewes. Little informa- exogenous MEL does not suppress the endogenous noc- tion is available with regard to effects of MEL on milk turnal secretion of MEL, as reported by O’Callaghan et composition. Protein content of milk increased (15%) al. (1991) and Malpaux et al. (1997) in sheep. in Polish Longwool ewes treated with MEL implants in Furthermore, MEL also controls the seasonal rhythm mid lactation under spring conditions, as reported by of prolactin (PRL) secretion by acting directly into the Molik et al. (2012). pituitary gland (Gómez-Brunet et al., 2008). The reduc- Based on the hypothesis that changes in MEL levels tion of MEL secretion during long days is accompanied may affect milk production and composition, the aim by an increase in PRL secretion in sheep (Lincoln and of this study was to evaluate the lactational effects of Clarke, 1994; Misztal et al., 1997). A reverse effect is subcutaneous MEL implants in 2 breeds of dairy sheep, observed during short days, where high MEL secretion basically differing in milk yield and milk composition inhibits PRL secretion and milk yield (Misztal et al., (major differences) and in their body weight (minor 2001; Molik et al., 2007). Dahl et al. (2000) concluded difference), in early lactation and under short-day pho- that milk production of dairy cows is negatively af- toperiod conditions. With this aim, the experimental fected by a short-day photoperiod, resulting in high period was centered in the winter solstice (minimum MEL, low PRL, and low IGF-I levels in blood. day length), when the expected secretion of endogenous The effects of MEL implants on lactational perfor- MEL should be maximum. mances of dairy small ruminants is nowadays a con- troversial topic, with results conditioned by day-length MATERIALS AND METHODS conditions. Thus, on one hand, Abecia et al. (2005) reported that the use of MEL implants in Lacaune and The study was conducted in the experimental farm Assaf dairy ewes during lactation in spring (long day) of the Servei de Granges i Camps Experimentals of the did not reduce milk yield. On the other hand, Molik et Universitat Autònoma de Barcelona in Bellaterra (Bar- al. (2010) applied MEL implants in Polish Longwool celona, Spain) located at N 41°30′20″ and E 2°05′46″ ewes in early lactation under long-day (i.e., spring) and during a 14-wk period, centered in the winter solstice short-day (i.e., autumn) conditions, using untreated (minimum day length, 9.25 h). Animal care conditions ewes under each photoperiod as control. The MEL- and management practices agreed with the Spanish treated ewes decreased PRL secretion, although the Royal Decree 53/2013 on the protection of animals used effect was only significant under long-day photoperiod. for experimental purposes, the codes of recommenda- The lowest PRL values were observed in both MEL tions for the welfare of dairy sheep of the Ministry of and control short-day ewes, and did not differ between Agriculture, Alimentation and Environment of Spain them. Moreover, the authors reported greater growth (MAPA, 2007), and the procedures stated by the Ethi- hormone in the plasma of long-day ewes and short-day cal Committee of Animal and Human Experimentation MEL-treated ewes, when compared with the other ewe of the Universitat Autònoma de Barcelona. groups. In later research, Misztal et al. (2018) revisited the Ewe Management and Feeding interaction of MEL and photoperiod in Polish Long- wool sheep, with the aim of mitigating the negative A total of 72 ewes of 2 dairy breeds (Manchega, MN, effects of late lactation in ewes lambing under long-day n = 36; Lacaune, LC, n = 36) in early lactation (35 photoperiod (June), when compared with those natu- ± 1 DIM) after the weaning of the lambs (28 DIM) rally lambing in short-day photoperiod (January). It were used. The ewes (MN vs. LC, respectively) dif- should be stressed, to avoid misunderstandings with the fered in BW (72.4 ± 1.9 vs. 77.7 ± 2.3 kg of BW; P = terminology previously used, that in this case milking 0.045) but had similar BCS (on average, 2.99 ± 0.12 (started after a long suckling) was consequently per- BCS units) and age (on average, 4.3 ± 0.4 yr). All formed mainly under short-day (autumn) and long-day ewes wore plastic ear tags (Allflex Europe) and ceramic (spring) photoperiods, respectively. Moreover, MEL rumen mini-boluses (20 g; Datamars) for visual and was applied 6 wk before and 6 wk after lambing (2 electronic identification, respectively, that were used doses with a 90-d interval). So, according to Misztal et for automatic milk recording as described by Ait-Saidi al. (2018), milk yield was greater in the long-day lactat- et al. (2014). ing ewes, in comparison with short-day (−20%) and Ewes were permanently sheltered on barley straw- short-day treated with MEL (−32%) lactating ewes. bedded pens and fed a TMR offered ad libitum. The Interestingly, plasma MEL mean concentration at late main axis of the shelter was east-west oriented and had pregnancy was inversely related to daylight duration an open wall with slats on the south wall, allowing Journal of Dairy Science Vol. 105 No. 3, 2022 Elhadi et al.: MELATONIN IN DAIRY EWES 2589 natural illumination and aeration. A supplementary using the milk-flow and milk recording automatic units program of artificial light for farm and milking manage- of the milking parlor. Data were uploaded using the ment (morning, 0600 to 1000 h; evening, 1800 to 2100 AlPro software 7.2 (DeLaval) and daily reviewed and h), providing 50 lx at the level of the ewes, was main- uploaded into a spreadsheet to avoid incorrect values. tained constantly during the experiment. The TMR Milk Composition. Representative milk samples of (forage:​concentrate, 60:40%; DM basis) consisted of each ewe were taken pre- (d −3) and posttreatment (d alfalfa hay as forage and a farm-produced concentrate 15, 30, 45, and 75), corresponding to 32, 50, 65, 80, and [ingredients: barley grain, 37.0%; corn grain, 15.0%; 110 DIM, for compositional analysis using proportional soybean hulls, 15.0%; gluten feed, 10.0%; soybean meal, milk samplers (MM25SG, DeLaval). Milk samples (50 5.0%; rapeseed 00 meal, 5.0%; oat grain, 4.5%; sunflow- mL) were composited according to the daily milking er meal, 2.0%; soybean oil, 2.0%; cane molasses, 2.0%; intervals (a.m.:p.m., 60:40), preserved with an antimi- dicalcium phosphate, 1.0%; calcium carbonate, 0.5%; crobial tablet (Bronopol, Broad Spectrum Micro-tabs Vitafac Ovino-0.3 premix (DSM Nutritional Products), II, D&F Control Systems) and stored at 4°C until 0.5%; and salt, 0.5%; as fed], on a DM basis. Moreover, analysis. Milk samples were analyzed in the Dairy Herd all ewes received 0.10 kg of corn whole grain at each Improvement Laboratory of Catalonia (ALLIC) for fat, milking, in the individual feeders of the milking parlor, total protein, lactose, and urea (Milkoscan FT2, Foss) to facilitate their entrance. Nutrient requirements were and SCC (Fossomatic 5000, Foss). based on INRA (2010) and calculated by INRAtion Feed Intake. Feed intake of each group was assessed v.4.07 (Educagri Éditions). Ewes had free access to wa- daily at the start (wk 2 and 3), mid (wk 6 and 7), and ter and commercial micromineral blocks (Multi-Block, final (wk 10 and 11) periods posttreatment by measur- Agrària Comarcal del Vallès). ing the amount of feed offered and refused in each pen of Milking machine started on d 29 after abrupt wean- 6 ewes. No refusals of concentrate were observed in the ing of the lambs. Milking procedures were like those milking parlor. The TMR offered in the pens and their described by Elhadi et al. (2019) and milking was con- refusals were sampled daily and composited weekly for ducted twice daily (0700 and 1700 h) in a 2 × 12-stall pre- and posttreatment periods feed analysis. parallel milking parlor (Amarre Azul I, DeLaval Equi- Feed Analyses. The DM content was determined pos) with a central high-milk pipeline, silicone milking by gravimetry, desiccating the sample in a forced-air clusters (SG-TF100, DeLaval), and automatic milk-flow stove (103°C for 24 h) and OM content was also mea- and milk recording devices (MM25SG, DeLaval). sured gravimetrically by ashing the samples in a muffle furnace (550°C for 4 h) according to AOAC (1990). Cel- Experimental Treatments lulose was analyzed as crude fiber according to AOAC (1990), and NDF and ADF were determined, on an After the weaning of the lambs (28 d of age), the ewes ash-free basis, by adding amylase and sodium sulfite were distributed in 12 balanced groups of 6 according solutions according to Van Soest et al. (1991) and us- to BW, BCS, age, milk yield, and milk composition. ing an Ankom200 Fiber Analyzer incubator (Ankom The experiment lasted 12 wk and consisted of a 1-wk Technology). Total N was determined by combustion adaptation period (28 to 35 DIM) and 11-wk post- according to the Dumas method using a Leco analyzer treatment period (36 to 112 DIM). The experimental (Leco Corporation), and CP was calculated as N × design consisted of a factorial with 2 treatments × 2 6.25. breeds × 3 replicates, to which the ewe groups were Blood Measures. Blood samples were taken from randomly allocated. Treatments were MEL (MN, n = the jugular vein using 10-mL Vacutainer tubes with 18; and LC, n = 18) consisting of a single MEL implant lithium heparin 170 IU (BD, Belliver Industrial Estate) (18 mg/ewe, Melovine, Ceva Animal Health), applied at 0800 h, after the morning milking and before feed- subcutaneously in the base of the ear at 35 ± 1 DIM; ing, at d −5 pre-treatment and d 15, 45, 75, and 89 and control (MN, n = 18; and LC, n = 18) that did posttreatment (corresponding to 30, 50, 80, 110, and not receive any treatment. At the treatment day, all 124 DIM). This sampling time was chosen to assess the ewes were restrained in the head locker of the feed the basal level of MEL with or without the implants. bunk after the p.m. milking and the MEL ewes were Plasma was obtained by whole blood centrifugation for implanted with a single implant in the left ear. 15 min at 2,000 × g and 4°C, transferred to 0.5-mL Eppendorf tubes, and stored at −20°C for analyses. Measurements, Sampling, and Analyses Hormone analyses in plasma were performed accord- ing to the ELISA sandwich type method by using Milk Yield. Milk yield of individual ewes was record- MEL (LDN), PRL (DIASource Immunoassays), and ed at each milking during the whole experiment (85 d), IGF-I (Mediagnost) kits. The stopped ELISA plates Journal of Dairy Science Vol. 105 No. 3, 2022 Elhadi et al.: MELATONIN IN DAIRY EWES 2590 were read in an automatic reader (iEMS Reader MF and the residual error. No significant interactions (P > V.2.9–0, Labsystems España) at 450 nm. Sensitivity 0.20) were removed from the model. Values of the vari- and intra- and interassay coefficients of variation were ables were computed for their respective treatment and MEL (1.0 ± 0.2 pg/mL, 8.5%, and 11.1%), PRL (0.35 sampling dates, the means expressed as least squares ± 0.07 ng/mL, 4.5%, and 5.9%), and IGF-I (0.09 ± means and separated by pairwise comparison using the 0.02 ng/mL, 8.0%, and 9.9%), respectively. PDIFF test of SAS. Pearson correlation coefficients (r) Body Reserves. The BW and BCS of all ewes were were calculated using the CORR procedure of SAS. evaluated 1 wk before the start of experiment, to allo- Significance was declared at P < 0.05 and a tendency cate the ewes in balanced groups during the adaptation was considered when P < 0.10. period, and at d 15, 30, 45, and 75 posttreatment (cor- responding to 28, 50, 65, and 80 DIM). Weighing was performed using an electronic scale (Tru-Test A6500), RESULTS AND DISCUSSION and BCS was assessed (0 to 5 points; accuracy, ±0.25 points) according to Russel et al. (1969). Although the dose of MEL (18 mg/ewe) was the same for the MN and LC ewes, because of the difference Statistical Analyses in BW between breeds (i.e., the LC ewes were 5.3 kg heavier than the MN), the mean MEL treatment dose Data were analyzed by the MIXED procedure for re- (0.240 mg/kg of BW) was 6.8% lower in the LC (0.232 peated measurements of SAS v.9.4 (SAS Institute Inc.). mg/kg of BW) compared with the MN (0.249 mg/kg The statistical mixed model included the breed (MN of BW) ewes. This source of variation has not been and LC), the melatonin treatment (MEL and control), considered in previous studies and should be considered the sampling time, and the breed × treatment and the in the analysis of our results and for further research. treatment × sampling interactions as fixed effects, as The main results of MEL treatment on the lactational well as the random effects of the experimental unit performances of the studied dairy ewes in early lacta- (ewe, for individual measurements, or pen for intake), tion are summarized in Table 1. Table 1. Effects of melatonin implants (18 mg/ewe) under short-day conditions of 2 breeds of dairy ewes in early lactation (LSM ± SEM) Manchega Lacaune Effect (P-value) Item CO1 MEL1 CO MEL Mean SEM Melatonin Breed Interaction2 Intake, kg of DM/d 2.26 2.19 3.37 3.29 2.78 0.07 0.32 0.001 0.96 Milk yield Yield, kg/d 1.43 1.32 2.54 2.51 1.95 0.15 0.67 0.001 0.80 ECM,3 kg/d 1.37 1.28 2.13 2.16 1.73 0.14 0.68 0.001 0.90 Fat, g/d 99 93 138 143 118 9 0.74 0.001 0.69 Protein, g/d 81 76 135 136 107 8 0.91 0.001 0.67 Lactose, g/d 70 64 121 120 93 8 0.84 0.001 0.77 TS, g/d 264 246 419 423 338 26 0.75 0.001 0.92 Milk content Fat, % 6.92 7.06 5.44 5.71 6.28 0.18 0.27 0.001 0.72 Protein, % 5.69 5.77 5.34 5.43 5.56 0.10 0.42 0.002 0.95 Lactose, % 4.87 4.83 4.76 4.77 4.81 0.08 0.84 0.30 0.75 TS, % 18.5 18.6 16.5 16.9 17.6 0.2 0.27 0.001 0.68 Urea, g/L 64.9 66.5 56.4 58.3 61.5 1.5 0.26 0.001 0.94 SCC, log10/mL 5.82x 5.30y 5.55 5.46 5.53 0.15 0.063 0.72 0.19 Plasma hormone Melatonin, pg/mL 6.88b 17.18a 5.75b 9.40a 9.80 1.05 0.001 0.001 0.002 Prolactin, ng/mL 19.8a 9.1b 13.2 11.1 13.3 3.4 0.050 0.49 0.19 IGF-I, ng/mL 258 283 272 294 276 27 0.41 0.66 0.97 Body reserves BW variation, kg/30 d 1.17 2.09 2.85 2.29 2.10 0.51 0.73 0.076 0.16 ADG, g/d 38.9 69.8 94.9 76.5 70.0 16.9 0.72 0.077 0.16 BCS variation in 30 d 0.22 0.13 0.08 0.10 0.13 0.06 0.52 0.18 0.33 a,b Within a row and breed, values with a different superscript differed (P < 0.05). x,y Within a row and breed, values with a different superscript tended to differ (P < 0.10). 1 CO = control; MEL = melatonin. 2 Melatonin × breed. 3 ECM = milk yield × [0.071 × (fat, %) + 0.043 × (total protein, %) + 0.2224], acco]rding to INRA (2010). Journal of Dairy Science Vol. 105 No. 3, 2022 Elhadi et al.: MELATONIN IN DAIRY EWES 2591 Feed Intake Our results agreed with those of Abecia et al. (2005) who reported no effects on milk yield throughout the Voluntary feed intake values of the dairy ewes are milking period of Lacaune and Assaf dairy ewes lambed shown in Table 1. No effects of the MEL implants on in autumn and winter, respectively, and treated with feed intake were detected in either breed (on average, MEL implants in spring (long-day) conditions. On the 2.78 ± 0.07 kg of DM/d; P = 0.32) during the 3 ex- contrary, Molik et al. (2013) in Polish Longwool ewes perimental periods studied [start (wk 2 and 3), mid milked after suckling in spring (long-day) conditions, (wk 6 and 7), and final (wk 10 and 11)] after MEL reported that MEL-treated and artificial short-day implantation. (200 lx light during 8-h) treated ewes yielded less milk Although, to our knowledge, the effect of MEL im- (−16% and −38%, respectively), than control ewes un- plants on feed intake in sheep is not well documented, der natural long-day photoperiod. Nevertheless, in this MEL alters leptin signaling in sheep with a greater study, MEL implants were applied twice during lacta- appetite-depressing effect of leptin being observed in tion (weaning, d 57; late lactation, d 147), although short natural day lengths (Adam and Mercer, 2004). the authors did not report the plasmatic MEL values. Song and Chen (2009) reporter greater plasmatic levels Bocquier et al. (1997) also reported that Sarda dairy of leptin and lower feed intake in water-supplemented ewes produced −25% milk under short-day photoperiod MEL mice. Bocquier et al. (1997) observed that DMI (light:​dark, 8:16) than those under long-day (16:8) con- of lactating Sarda dairy ewes under the short-day pho- ditions during lactation. toperiod treatment was lower (−16%) than ewes under Misztal et al. (2018) reported that milk yield of Pol- long days. Lacasse et al. (2014) reported a 4% decrease ish Longwool sheep ewes, implanted twice with MEL of DMI in Holstein cows fed MEL and under long-day (in late pregnancy in spring and in early lactation in photoperiod, when compared with those under short- autumn), or left as control under the same photoperiod day photoperiod conditions. The effect of photoperiod conditions, was greater in the long-day lactating ewes, length on feed intake was not significant in lactating when compared with the control in short-day (−20%) Saanen dairy goats under short- and long-day photope- and the MEL treated in short-day (−32%) conditions. riod conditions (Mabjeesh et al., 2007). Consequently, Despite the species differences, the use of MEL during according to these data, a slight decrease or no effect in late pregnancy (2 implants of 18 mg/goat) was also DMI should be expected when dairy ewes are treated studied by Avilés et al. (2019) in Creole goats dried off with MEL implants during lactation. during the summer solstice that kidded in late August The effect of the breed on DMI was important in and were suckled and milked during autumn (short day). our results, and on average, MN ewes ate 33% less DM The MEL treatment, removed surgically at kidding, than did LC ewes (P < 0.001; Table 1). This result stimulated the subsequent lactational performances and agrees with the greater milk yield of the LC ewes (P increased milk yield more than 20% during suckling (as < 0.001), as later discussed, and with the results of well as kid growth) and milking, but milk composition previous studies comparing both breeds in the same was not affected. This increase in milk yield agreed with stage of lactation (Molina et al., 2001; Flores et al., those reported in Holstein cows (Auchtung et al., 2005) 2008; Elhadi et al., 2019). No MEL × breed interaction and Saanen goats (Mabjeesh et al., 2013) exposed to was detected on the feed intake of our ewes (P = 0.96; artificial short days (light:​dark, 8:16 h) during the dry Table 1). period, which produced more milk in the subsequent lactation under natural long-day photoperiod. It should Milk Yield be stressed that in the Mabjeesh et al. (2013) experi- ment the goats were under hot weather conditions in No differences by effect of MEL treatment were de- late pregnancy, which is known to dramatically increase tected on milk yield (P = 0.67) and ECM (P = 0.68) PRL levels, as later discussed. Nevertheless, Dahl et al. in both breeds (Figure 1). No MEL × breed interaction (2000) and Lacasse et al. (2014) reported that feeding was detected on milk yield or milk components yield MEL during late pregnancy had no effects on the sub- of our ewes (P = 0.67 to 0.92) as shown in Table 1 sequent milk yield of Holstein dairy cows, but the result and Figure 1. On average, and for the same stage of may have been conditioned to the MEL source. Thus, lactation, LC ewes yielded 83% more milk than MN Auldist et al. (2007) evaluated the effects of repeated ewes did (LC vs. MN, 2.53 ± 0.15 vs. 1.38 ± 0.15 kg/d; MEL implants during 12 wk (6 × 18 mg/cow) in twin P < 0.001) throughout the experiment. The lactation New Zealand Friesian dairy cows calved in spring. The persistency linear coefficients in early lactation (35 to use of MEL implants decreased milk yield (−23%) and 95 DIM) were similar for the MN and LC ewes (−11 increased milk contents, as in late lactation. Auldist and −14 g/d, respectively; P = 0.83). et al. (2007) concluded that some of the milk changes Journal of Dairy Science Vol. 105 No. 3, 2022 Elhadi et al.: MELATONIN IN DAIRY EWES 2592 observed in New Zealand throughout the summer may goats during late pregnancy (Avilés et al., 2019) did be a consequence of the MEL increase associated with not change milk composition and the use of short-day the decreasing day length. photoperiod during late pregnancy in Saanen dairy goats did not change their milk composition (Mabjeesh Milk Composition et al., 2007). With regard to dairy cows treated with MEL implants during summer in New Zealand, Auldist No changes on milk components by effect of MEL et al. (2007) reported increases in milk fat and protein treatment, under short-day conditions (winter solstice), contents (14% and 7%, respectively), whereas lactose were detected in our ewes throughout the experiment, content decreased (−7%), but no effects were detected as well as on the daily yield of different milk components by Lacasse et al. (2014) when the milk of short-day, (P = 0.25 to 0.99; Table 1). On the other hand, marked long-day, and MEL-fed lactating Holstein cows under differences in milk fat and milk protein contents were long-day photoperiod was compared. observed between MN and LC breeds (Figure 2), but not in their lactose content (P = 0.84), as previously re- Plasmatic Hormones ported for the 2 breeds under similar conditions (Flores et al., 2008; Elhadi et al., 2019). Only SCC tended to Hormonal values in the plasma of our ewes according decrease by effect of the MEL treatment in the case of to treatments and breed are summarized in Table 1 MN ewes (P = 0.063). and changes of basal MEL through the experiment are On the contrary, Molik et al. (2012) reported that shown in Figure 3. Plasmatic levels of MEL in sheep the administration of MEL increased the protein con- are highly variable among individuals (i.e., 0 to 300 tent (15%) of the milk of Polish Longwool ewes under pg/mL), but the nocturnal MEL concentration is high long-day photoperiod. Moreover, Bocquier et al. (1997), (>100 pg/mL) and characteristic of each individual, in Sarda, and Molik et al. (2012), in Polish Longwool which is indicative of a genetic control (Chemineau et ewes, also reported the positive effects of short days al., 1996; Zarazaga et al., 1998). On the contrary, MEL versus long days on milk protein content (4% and 15%, levels are low (i.e., 0 to 10 pg/mL) and stable after the respectively). Nevertheless, implanting MEL to Creole dawn (O’Callaghan et al., 1991). Because the pineal Figure 1. Effects of a single s.c. melatonin implant (MEL; 18 mg/ewe, d 35) on milk yield of Manchega (□, MN-control; ■, MN-MEL) and Lacaune (○, LC-control; ●, LC-MEL) dairy ewes in early lactation (35 DIM) under autumn photoperiod conditions. Treatments: melatonin (solid lines and closed symbols, ■ and ●) or control (broken lines and open symbols, □ and ○). Values are LSM with the SEM indicated by vertical bars. Journal of Dairy Science Vol. 105 No. 3, 2022 Elhadi et al.: MELATONIN IN DAIRY EWES 2593 MEL (central) is mainly secreted during the night and implants were applied, represented the total MEL con- its concentration falls after dawn, values obtained from centration in blood (endogenous-peripheral secretion blood samples collected from the control ewes after the plus exogenous). a.m. milking represented, according to Kvetnoy (1999), As shown in Figure 3, no changes in the basal MEL the basal endogenous extrapineal MEL secretion (pe- values were observed in the control ewes throughout ripheral) during the short-day photoperiod conditions the experiment (6.4 ± 1.2 pg/mL, on average; Table 1), of our experiment. On the other hand, the values of although MN showed numerically greater basal MEL the MEL-treated ewes after d 35, when the slow-release values than LC ewes. After the insertion of implants (18 mg/ewe), MEL values in plasma increased mark- edly in the treated ewes, but this increase was greater in MN than in LC ewes (150 vs. 63%, respectively; P < 0.001). Such differences were not explained by the practical MEL doses reported in our ewes according to breed BW (MN, 0.249 mg/kg of BW; LC, 0.232 mg/kg of BW) and should be explored by further research. Plasmatic MEL values in the treated ewes reached a plateau of at least 60 d (d 50 to 110, Figure 3) and decreased to basal values thereafter (d 125). The difference between breeds was unexpected and we hypothesized that it may be a consequence of differ- ences in the metabolization of exogenous MEL, which could be associated with the greater metabolic activity of the higher yielding LC ewes, as well as with the greater BW and BCS (Table 1) observed in MN than in LC ewes. Melatonin is recognized by its functional synergism with insulin, decreasing glycemia and insulin resistance (Cipolla-Neto et al., 2014). After the discovery of association of MEL receptor gene MTNR1A (MT1) polymorphisms with sheep fertility (Notter and Chemineau, 2001; Notter et al., 2003), Mura et al. (2010) reported stronger effects of exogenous MEL on the reproductive activity of Sarda dairy ewes homozygous to the MT1 favorable allele (+/+). This would also explain the link between geno- type and photoperiod, as the −/− ewes need higher and long-lasting levels of MEL in blood to be stimulated for reproduction. It should be stressed that, in our MN and LC experimental flock, adult ewe and hogget fertility during spring was repeatedly greater in LC than in MN sheep (data not shown). Consequently, the use of a unique dose of MEL recommended in practice (a single implant of 18 mg/ ewe) may be questioned and further study is needed to determine if it is optimal for heavier and high-yielding dairy sheep breeds (e.g., Assaf and East Friesian ewes). Misztal et al. (2018) reported dramatic increases of nocturnal blood MEL in Polish Longwool ewes when compared short-day photoperiod (400%) or MEL im- Figure 2. Effects of a single s.c. melatonin implant (MEL; 18 planted under long-day conditions (425%), with long- mg/ewe, d 35) on milk composition (a, fat; b, protein; c, lactose) of Manchega (□, MN-control; ■, MN-MEL) and Lacaune (○, LC-control; day photoperiod as control. The increases were greater ●, LC-MEL) dairy ewes in early lactation (35 DIM) under autumn than in our results as a consequence of sampling time, photoperiod conditions. Treatments: melatonin (solid lines and closed which was after the sunset in Misztal et al. (2018) and symbols, ■ and ●) or control (broken lines and open symbols, □ and ○). Values are LSM of both breeds averaged, with the SEM indicated included the nocturnal MEL secretion, whereas it was by vertical bars. after the dawn (i.e., only basal) in our experiment. Journal of Dairy Science Vol. 105 No. 3, 2022 Elhadi et al.: MELATONIN IN DAIRY EWES 2594 In agreement with the greater plasmatic MEL level of compared with the control ones. In the Mabjeesh et al. the MN-treated ewes, plasma concentration of PRL de- (2013) experiment, all of the goats were submitted to creased (−54%) when compared with MN-control ewes heat-stress in late pregnancy, which is known to dra- (Table 1). No effects between treatments were detected matically increase PRL levels (Hooley et al., 1979). The in the LC ewes. Molik et al. (2013) and Misztal et al. positive effects of short days during pregnancy on milk (2018) also reported decreases in plasma PRL concentra- yield were attributed to the inhibitory effects of MEL tion of the MEL-treated Polish Longwool ewes (−22%). on PRL secretion, which stimulated the involution of According to Misztal et al. (2018), plasma PRL in the the mammary gland and increased the expression of long-day ewes increased from late pregnancy (winter) PRL-receptors in different tissues (Auchtung et al., to weaning (spring), whereas in the short-day and the 2005), such as in the mammary gland and liver. Thus, short-day MEL-treated ewes the direction of change the higher the plasma MEL concentration, the lower was the opposite, with the lowest PRL concentration the PRL concentration in blood, and vice versa, such occurring at weaning (August). as observed in our MN and LC ewes. The decreasing effect of exogenous MEL on PRL No changes in plasma IGF-I values by effect of MEL secretion was also observed during increasing photope- treatment were detected in our ewes of either breed riod conditions, despite the strong stimulation induced (P = 0.66; Table 1). Nevertheless, Mabjeesh et al. by the suckling of the lambs (Molik et al., 2010). A (2007) reported a 68% decrease in plasma IGF-I values marked PRL decrease (−46%) was also observed in of short-day photoperiod lactating Saanen dairy goats lactating Saanen dairy goats under a short-day pho- and Auldist et al. (2007) in MEL-treated cows reported toperiod (Mabjeesh et al., 2007) and in MEL-treated a decrease in PRL plasmatic concentrations, but values New Zealand Friesian dairy cows (Auldist et al., 2007), of IGF-1 did not change. Figure 3. Effects of a single s.c. melatonin implant (MEL; 18 mg/ewe, d 35) on plasma melatonin of Manchega (□, MN-control; ■, MN- MEL) and Lacaune (○, LC-control; ●, LC-MEL) dairy ewes in early lactation under autumn photoperiod conditions. Treatments: melatonin (solid lines and closed symbols, ■ and ●) or control (broken lines and open symbols, □ and ○). Gray area and dotted line show the daylight duration. Values are LSM with the SEM indicated by vertical bars. Journal of Dairy Science Vol. 105 No. 3, 2022 Elhadi et al.: MELATONIN IN DAIRY EWES 2595 Body Reserves Ait-Saidi, A., G. Caja, A. A. K. Salama, and S. Carné. 2014. Im- plementing electronic identification for performance recording in sheep: I. Manual versus semiautomatic and automatic recording The BW and BCS of our ewes are shown in Table systems in dairy and meat farms. J. Dairy Sci. 97:7505–7514. 1. Despite the previously mentioned effects of MEL on https:​/​/​doi​.org/​10​.3168/​jds​.2014​-8090. leptin signaling, MEL treatment had no effects on BW AOAC. 1990. Official Methods of Analysis of the Association of Of- ficial Analytical Chemists. 15th ed. Association of Official Analyti- or BCS (P = 0.30 to 0.79) of the ewes of either breed. cal Chemists. On average, BW and BCS were 77.9 ± 1.1 kg and 3.02 Auchtung, T. L., A. G. Rius, P. E. Kendall, T. B. McFadden, and ± 0.05 units for early lactation under short-day photo- G. E. Dahl. 2005. Effects of photoperiod during the dry period on prolactin, prolactin receptor, and milk production of dairy period conditions. Bocquier et al. (1997) also found no cows. J. Dairy Sci. 88:121–127. https:​/​/​doi​.org/​10​.3168/​jds​.S0022​ effect of photoperiod length on body reserves of lactat- -0302(05)72669​-2. ing Sarda dairy ewes. No differences were found within Auldist, M. J., S. A. Turner, C. D. McMahon, and C. G. Prosser. 2007. Effects of melatonin on the yield and composition of milk from and between the short-day, long-day, and the MEL grazing dairy cows in New Zealand. J. Dairy Res. 74:52–57. https:​ with long-day treatment in lactating Polish Longwool /​/​doi​.org/​10​.1017/​S0022029906002160. ewes (Misztal et al., 2018). Avilés, R., J. A. Delgadillo, J. A. Flores, G. Duarte, J. Vielma, M. J. Flores, K. Petrovski, L. A. Zarazaga, and H. Hernández. 2019. Melatonin administration during the dry period stimulates subse- CONCLUSIONS quent milk yield and weight gain of offspring in subtropical does kidding in summer. J. Dairy Sci. 102:11536–11543. https:​/​/​doi​ .org/​10​.3168/​jds​.2019​-16836. The use of exogenous MEL, together with the effect Bittman, E. L., F. J. Karsch, and J. W. Hopkins. 1983. Role of pineal of the endogenous MEL under decreasing photoperiod gland in ovine photoperiodism: regulation of seasonal breeding and conditions, had no significant effects on milk yield and negative feedback effects of estradiol upon luteinizing hormone secretion. Endocrinology 113:329–336. https:​/​/​doi​.org/​10​.1210/​ composition of medium- and high-yielding dairy ewes endo​-113​-1​-329. in early lactation. Plasmatic PRL tended to decrease Bocquier, F., S. Ligios, G. Molle, and S. Casu. 1997. Effet de la photo- in the MEL-treated ewes, although the effect was only période sur la production, la composition du lait et sur les consom- mations volontaires chez la brebis laitière. Ann. Zootech. 46:427– significant in the MN ewes. On the other hand, marked 438. https:​/​/​doi​.org/​10​.1051/​animres:​19970505. differences in MEL plasmatic values were observed in Borjigin, J., X. Li, and S. H. Snyder. 1999. The pineal gland and the response to the same dose of MEL implants (18 melatonin: Molecular and pharmacologic regulation. Annu. Rev. Pharmacol. Toxicol. 39:53–65. https:​/​/​doi​.org/​10​.1146/​annurev​ mg/ewe) between breeds, the effect of the dose being .pharmtox​.39​.1​.53. greater in the MN ewes, which needs further research. Chemineau, P., I. Beltrán de Heredia, A. Daveau, and L. Bodin. 1996. High repeatability of the amplitude and duration of the nycthem- eral rhythm of the plasma melatonin concentrations in the Ile-de- ACKNOWLEDGMENTS France ewes. J. Pineal Res. 21:1–6. https:​/​/​doi​.org/​10​.1111/​j​.1600​ -079X​.1996​.tb00264​.x. This study was partially funded by Ceva Animal Cipolla-Neto, J., F. G. Amaral, S. C. Afeche, D. X. Tan, and R. J. Re- iter. 2014. Melatonin, energy metabolism, and obesity: A review. J. Health (Barcelona, Spain). The authors are grateful to Pineal Res. 56:371–381. https:​/​/​doi​.org/​10​.1111/​jpi​.12137. Alex Martino, Alessio Valenza, and Juan-Pedro Casas Dahl, G. E., B. A. Buchanan, and H. A. Tucker. 2000. Photoperiodic from Ceva Animal Health for their advice and sup- effects on dairy cattle: A review. J. Dairy Sci. 83:885–893. https:​/​ /​doi​.org/​10​.3168/​jds​.S0022​-0302(00)74952​-6. port. Thanks are also extended to Ramón Costa and Elhadi, A., A. A. K. Salama, X. Such, E. Albanell, P. G. Toral, G. the technical team of the SGCE (Servei de Granges i Hervás, P. Frutos, and G. Caja. 2019. Effects of shearing 2 breeds Camps Experimentals) of the Universitat Autònoma de of dairy ewes during lactation under mild winter conditions. J. Dairy Sci. 102:1712–1724. https:​/​/​doi​.org/​10​.3168/​jds​.2018​-15380. Barcelona (Bellaterra, Barcelona, Spain) for the care of Flores, C., G. Caja, R. Casals, E. Albanell, and X. Such. 2008. 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