Graduate Student Literature Review: The challenge of drying-off high-yielding dairy cows*

The cessation of lactation (i.e., dry-off) in dairy cattle is an area of research that has received much focus in recent years. The dry period is necessary to optimize tissue remodeling of the mammary gland, but represents a stressful event, incorporating several changes in daily routine, diet, and metabolism. Moreover, the high milk yields achieved by modern cows in late gestation exacerbate the need for relevant manipulations in the days around dry-off, as excessive accumulation of milk might jeopardize the success of the dry period, with potential negative effects on future lactation. Production levels over 15 kg/d are an additional risk factor for udder health, delay mammary involution, and worsen metabolic stress and inflammatory responses. Furthermore, the pressure to reduce antibiotic usage in farm animals has resulted in increased attention on the dry period, given that historically most dairy cattle were provided prophylactic intramammary antibiotic treatment at dry-off as a means to reduce the risk of intramammary infections in the subsequent lactation. Several strategies have been proposed over the years to cope with these challenges, aiming to gradually reduce milk yield before dry-off, promoting at the same time the start of mammary involution. Among them, the most common are based on feed or nutrient restriction, a decrease in milking frequency, or administration of prolactin inhibitors. These practices have different capacities to reduce milk yield through different mechanisms and entail several implications for udder health, animal welfare, behavior, endocrine status, metabolism, and inflammatory conditions. The present review aims to provide a comprehensive overview of the dry-off phase in high-yielding cows and of the impact of high milk production at dry-off, and to describe possible strategies that might be implemented by farmers and veterinarians to optimize this critical phase in an integrated way.


INTRODUCTION
The dry period, which is the nonlactating period between dry-off and calving, represents an important phase of the cow's production cycle, with relevant implications for cows' health and physiology, animal welfare, and sustainability of dairy production.The dry-off phase begins with the cessation of milking at the end of lactation to prepare the cow's metabolism and the udder for the maximization of milk production in the following lactation.During this period, cows complete the restoration of body condition and renew mammary epithelial cells (Capuco et al., 1997).The mammary gland initially undergoes a process of active involution, where, through a series of coordinated changes in the mammary gland morphology, the integrity of tight junctions, and the composition of mammary secretions, returns to a nonlactating state (Zhao et al., 2019).Traditionally, cows were dried off on a scheduled day, about 6 to 8 wk before expected calving, through abrupt cessation of milking and administration of longacting, intramammary antibiotic treatment.Thus, the dry-off event is the start of a period where the cow's physiology adapts initially to a nonlactating state but then prepares for the approaching parturition event.
In recent years, milk yield per cow has dramatically increased, and it is becoming increasingly common to find cows that still have high production (i.e., >20-25 kg/d) in late gestation (Zobel et al., 2015).Therefore, the transition from lactation to a nonlactating state is even more challenging.

Graduate Student Literature Review: The challenge of drying-off high-yielding dairy cows*
Blanket dry cow therapy (DCT) consists of the systematic administration of antimicrobials to all quarters of all cows in the herd at the time of dry-off to prevent and treat intramammary infections (IMI) during the dry period (Neave et al., 1969), which is, in particular at its beginning and end, the time at highest risk of IMI (Bradley and Green, 2004).In many countries, it is the most widespread approach (McCubbin et al., 2022).However, antimicrobial resistance has become a relevant concern for animals but also for possible implications for human health.Reducing antimicrobial usage in livestock has the potential to reduce the prevalence of antimicrobial resistance and to improve sustainability of dairy products (Tijs et al., 2022).Consequently, blanket DCT has been questioned and selective DCT, consisting of only treating with antibiotics subjects infected or at higher risk of developing IMI during the dry period, is now considered mandatory by many jurisdictions.For instance, Nordic countries has promoted similar approaches since 2009 (Rajala-Schultz et al., 2021), and the Netherlands banned the prophylactic use of antibiotics in livestock in 2012 (Vanhoudt et al., 2018), followed by the whole European Union in 2022.Therefore, selective DCT is becoming widespread with favorable results in antibiotic use reduction without major side effects in terms of increased SCC and mastitis incidence in the subsequent lactation (Kabera et al., 2021;Krattley-Roodenburg et al., 2021).Nevertheless, drying-off cows still producing a huge amount of milk without the support of antibiotic action might represent a threat to IMI and a risk for animal health or welfare.
To cope with this multifaceted challenge, several strategies were proposed, and literature about milk cessation methods, selective DCT, nutritional interventions, and their implications on animal physiology, metabolism, health, and future lactations is rapidly growing.However, available literature should be harmonized to provide an integrated approach to dry-off management.Therefore, this literature review summarizes the latest research on the different management practices associated with the dry-off period, with specific focus on those cows producing high volumes of milk at the time that dry-off is initiated.Then, the present review describes possible strategies that could be adopted on farms that could potentially improve cow health and reduce distress, with a perspective on the outcomes in the subsequent lactation.

THE PHYSIOLOGY OF DRY-OFF
On the majority of farms in the world cows are abruptly dried off [i.e., around 75% of farms in the United States and Europe (De Prado-Taranilla et al., 2020;Vilar and Rajala-Schultz, 2020)], meaning that milking stops immediately.This process is often combined with sudden diet and group changes (Zobel et al., 2015), making the dry-off a stressful event for dairy cows.Therefore, cows implement a series of physiological and morphological mechanisms to adapt to new conditions.Among them, one of the most important is the active involution of the mammary gland, which starts within 2 d after dry-off and ends in about 3 wk (Hurley, 1989), and allows the secretory tissue remodeling (Zhao et al., 2019).Moreover, the energy density of the diet is reduced, increasing forage inclusion in the ration, to foster the regression of galactopoietic activity and match the lower requirements of the dry period.In this phase, the fetus becomes the priority over the mammary gland (Dingwell et al., 2001).At the same time, rumen papillae decrease in length and size to adapt to the more fibrous and less digestible diet, and rumen bacterial community changes (Dieho et al., 2016(Dieho et al., , 2017)).The regrouping and the need to establish a new social structure could add psychological stress, particularly in weaker and subordinate animals (von Keyserlingk et al., 2008).Common markers of stress, such as blood cortisol and fecal glucocorticoids, increase after dry-off (Bertulat et al., 2013;Putman et al., 2018) and this condition can have different duration based on the ability to adapt to the previous changes.
The end of milk removal leads to accumulation of milk in the udder, a consequent engorgement of cisternal ducts and alveoli, and an increase in intramammary pressure, which trigger the involution process (Wilde et al., 1997).The prolactin signals previously associated with each milking stop when cows are no longer milked, promoting apoptosis of mammary epithelial cells.Further, residual milk and local hormonal release can participate in the inhibition of milk synthesis, with β-CN and serotonin being 2 main candidates for this role (Collier et al., 2012;Shoshani and van Straten, 2022).Mammary epithelium tight junction permeability increases, resulting in an exchange of components between milk and interstitial fluid (Stelwagen et al., 1994).Mammary secretion composition changes, with higher levels of SCC and components of blood origin (e.g., lactoferrin, immunoglobulins, albumin), altered ion concentration, and decreased citrate (Zhao et al., 2019).Conversely, substances of milk origin, such as lactose, are increased in the bloodstream.Thus, variations in these parameters can be used as markers of involution.In the first stages of involution, epithelial cells undergo programmed cell death and neutrophils start to infiltrate the gland (Atabai et al., 2007).The initial clearance of apoptotic cells and milk fat globules is mainly performed by viable mammary epithelial cells, given the limited presence of professional phagocytes at this stage.Later, epithelial cell apoptosis increases, the alveolar luminal structure collapses, and macrophages and lymphocytes infiltrate the gland to phagocytize dead cells and clear milk debris (Atabai et al., 2007).Therefore, the immune system is likely challenged by the removal of milk components and apoptotic cells and perhaps less active in preventing pathogen infiltration.
High pressure and udder engorgement impair the teat canal closure process and the formation of the protective keratin plug on the teat sphincter, increasing the risk of milk leakage (Dingwell et al., 2001).Because cows suffering from milk leakage at dry-off have an increased probability of pathogen entrance (Dingwell et al., 2004), the latter becomes a risk factor for the development of new IMI in the first month of lactation (De Prado-Taranilla et al., 2020).After completion of involution, the combination of morphological, physiological, and immune modifications drastically reduces the risk of IMI (Bradley and Green, 2004).In particular, the antimicrobial activity of lactoferrin, immunoglobulins, leukocytes, and milk pH contribute to creating a hostile environment for bacterial growth.
The effects of the several changes and the involution process can be observed in blood (Putman et al., 2018;Mezzetti et al., 2020).Following dry-off, metabolic adaptations occur and the immune system can be overwhelmed in this phase by the variety of functions it must serve (i.e., removal of residual milk and apoptotic cells, defense from the potential entrance of pathogens, systemic activation).Plasma NEFA increase after dryoff due to the drop in energy and nutrient intake, and urea concentrations decrease as a result of the changes in mammary gland requirements, feed intake, and rumen fermentation.Nevertheless, the effects on glucose and BHB are inconsistent, likely depending on milk yield at dry-off and the magnitude of diet change.Concurrently, the need for the clearance of milk residuals and mammary tissue remodeling trigger an inflammatory and immune response (Putman et al., 2018).After dry-off, leukocytes migrate toward the mammary gland, reducing their concentration in the bloodstream (Sordillo and Nickerson, 1988;Atabai et al., 2007), and their activation could account for the release of pro-inflammatory stimuli (i.e., cytokines) into the blood and the consequent inflammatory response taking place.This inflammatory response is usually mild, if there are not ongoing IMI, and can be observed as an increase in haptoglobin and serum amyloid A levels, which can be related to the process of tissue remodeling (Odensten et al., 2007;Dancy et al., 2019).Thus, liver function markers (γ-glutamyl transferase and bilirubin), positive acute-phase proteins (ceruloplasmin and serum amyloid A), and nitrogen species (nitrate, nitrite, and nitric oxide) levels increase, whereas negative acute-phase pro-teins (cholesterol and retinol) concentrations decrease (Mezzetti et al., 2020).Antioxidant species (i.e., ferric reducing antioxidant power, thiol groups, tocopherol, and β-carotene) decrease their blood concentrations as a result of the inflammatory response and the intense degradation of milk residuals in the mammary gland immediately after milking cessation.Moreover, blood calcium greatly increases as a consequence of either reduced milk demand or milk accumulation in the mammary gland, which compromises mammary tight junction integrity, increasing paracellular transport of calcium into the blood (Putman et al., 2018;Mezzetti et al., 2020).The dry-off is thus a stressful phase for cows, with drastic and intense changes, that take place mainly at the mammary, rumen, and liver levels.Considering all the intense variations, the potential concerns and difficulties of dry-off and subsequent dry period, and to avoid repeated transitions, the usefulness of milking cessation and dry period has been questioned.

THE IMPORTANCE OF THE DRY PERIOD AND ITS OPTIMAL LENGTH
In natural conditions, the dam gradually reduces milk production (i.e., gradual involution) in response to the lowering demand of the offspring and the reduced suckling frequency (Vilar and Rajala-Schultz, 2020).In commercial settings, cows still produce large amounts of milk in late gestation and removal of milk is often stopped abruptly, causing the alterations described in the previous section.To mitigate these effects, one possible solution to is to omit or shorten the dry period.The main aim of this strategy is to improve energy balance in early lactation shifting milk production from the postcalving period to the last weeks of gestation (van Knegsel et al., 2013).Additionally, cows have to face fewer dietary and group changes.Andersen et al. (2005) compared a 7-wk dry period with continuous milking through late gestation in cows with peak milk yield greater than 45 kg/d.These authors reported a consistent decrease in milk production in the following early lactation, but a mitigated negative energy balance in cows that skipped the dry period.Alternatively, shortening the dry period (up to about 30 d) can potentially balance the side effects of the different dry period strategies.Compared with a 6 to 8 wk nonlactating period, a short dry period resulted in a slight milk loss in the subsequent lactation (-4.5%), but improved milk protein and energy balance (van Knegsel et al., 2013).A short dry period also seems to improve peripartum energy balance and ruminal adaptation compared with a 60-d dry period (Jolicoeur et al., 2014).Various hypotheses have been proposed to explain the milk loss caused by shortening dry period length but the most likely is related to the effects of dry period on cell turnover and replacement of senescent mammary epithelial cells (Capuco et al., 1997).This process is completed in approximately 25 d (Capuco et al., 1997), which thus represents the minimum length requirement of dry period.However, short or omitted dry period can also influence udder health.Shortening the dry period did not seem to affect udder health, with no negative effects on the odds of mastitis (van Knegsel et al., 2013).However, most of the trials available in the literature were carried out using blanket DCT, which could have affected the outcomes (Kok et al., 2019).Cows selectively treated at dry-off might benefit from a longer dry period.In fact, the dry period is an important phase for curing IMI, even subclinical (i.e., low level of SCC but presence of some strains of pathogens) because, alongside the antibiotic effect, there is the physiological clearing of many bacteria during the mid-dry period.

THE EFFECT OF HIGH MILK YIELD AT DRY-OFF
Over the past decades, continuous improvements in management, nutrition, and genetics allowed for greatly improve milk yield and lactation persistence.However, one possible side effect of these advances is the high level of production at dry-off (increased 2-3 times in the last 4-5 decades; Zobel et al., 2015), which makes the transition to the dry period challenging.The need to reduce milk production before stopping milking was identified as important at least 70 yr ago.In those times, gradual dry-off was performed to reduce the risk of mastitis caused by high intramammary pressure (Oliver et al., 1956), but, with the spread of antibiotic therapy at dryoff, it was gradually replaced by abrupt dry-off (Neave et al., 1969).However, milk yields at dry-off were below 10 kg/d (Natzke et al., 1975).Around 20 yr ago, Dingwell et al. (2001) returned to the question.Nowadays, production at dry-off can easily exceed 25 kg/d and application of selective DCT is steadily increasing.The effect of an abrupt dry-off performed without antibiotic therapy on a cow producing a large amount of milk is unknown, but this practice is typically avoided to preserve animal health and welfare.High milk yield at dry-off is one of the main reasons that leads farmers to use antibiotic DCT in Austria and the Netherlands, even in cows with low SCC (Wittek et al., 2018;Krattley-Roodenburg et al., 2021).Nevertheless, to our knowledge, no study has specifically investigated the effects of high milk yield at the final milking in cows not receiving antibiotic DCT.In a recent study, antibiotic use, udder health, and milk yield have been reported to not be markedly affected by selective DCT compared with blanket DCT, regardless of milk production level at the last herd test (Rowe et al., 2023).However, in another observational study, the odds of clinical masthan 14 kg/d had a smoother transition into the dry period (Silanikove et al., 2013).In another study, cows with milk yield greater than 15 kg/d during the week preceding dry-off have more severe metabolic changes and an increased and longer inflammatory response around dry-off compared with low-producing subjects (Mezzetti et al., 2020).Excessive milk accumulation in the udder after milking cessation can also provoke pain or discomfort associated with the engorged udder, as suggested by the reduced lying time observed in cows with high milk yield at dry-off (Rajala-Schultz et al., 2018).Therefore, strategies to reduce milk yield at dryoff and its negative effects are needed.

APPROACHING THE DRY-OFF EVENT: HOW TO REDUCE MILK PRODUCTION?
Abrupt cessation of milking is a practical solution in commercial farms but may represent a threat to udder health and animal welfare in cows still producing a generous amount of milk (Zobel et al., 2015;Vilar and Rajala-Schultz, 2020).Thus, to ensure udder health, promote mammary involution, and optimize behavioral response to dry-off, a target of about 15 kg/d has been suggested (Vilar and Rajala-Schultz, 2020).Different drying-off practices have been proposed to gradually reduce milk yield in late lactation.These methods mainly include gradual milking frequency reduction, diet changes, or a combination.The main effects of these approaches are summarized in Table 1.The greatest challenge is to develop a gradual cessation method able to reduce milk production at safe levels without adversely affecting cow metabolism, immune system function, and welfare (Zobel et al., 2015).Gradual dry-off strategies are common in countries where selective DCT is mandatory (Vilar et al., 2018;Krattley-Roodenburg et al., 2021) but not where blanket DCT is prevalent (Bertulat et al., 2015), indicating the need for systems to reduce milk yield when antibiotic use has to be limited.
Especially in selective DCT, the blanket use of internal teat sealant is recommended (Kabera et al., 2021).The function of teat sealant is to close the teat canal immediately after dry-off, preventing the entrance of bacteria.Its use at dry-off has a protective effect on the development of IMI, both alone and in combination with antibiotic treatment (McParland et al., 2019).In high-yielding cows, teat sealants might be even more beneficial.These animals have higher intramammary pressure, delayed formation of the keratin plug, increased probability of milk leakage (Dingwell et al., 2004;De Prado-Taranilla et al., 2020), and, thus, more favorable conditions for the development of IMI (Dingwell et al., 2001), that could be ameliorated by teat sealant application.Although Godden et al. (2003) did not find any interaction between internal teat sealant effectiveness and milk yield at dry-off, new studies evaluating this relationship might be useful for producers and veterinarians.
Milking can be stopped abruptly or gradually (Vilar and Rajala-Schultz, 2020).Gradual milk cessation can be performed by reduced milking frequency or intermittent milking.The first strategy consists of switching from the usual 3× or 2× daily milkings schedule to 1× around a week before dry-off or can also be applied through intermittent milking (e.g., 1× milking/d on d 1, 2, 3, and 5 and then dry-off at d 5).Through this strategy is possible to achieve up to a 40% reduction in milk yield (Figure 1).The reduction in milking frequency inhibits milk synthesis and promotes apoptosis by local stimuli, due to prolonged milk accumulation in the udder between milkings (Wilde et al., 1997).The main concern about this practice is the increase in intramammary pressure, which can affect cow comfort and result in milk leakage (Tucker et al., 2007).The latter is a risk factor for IMI.Gott et al. (2016) reported that 8.0% of quarters and 14.3% of cows were observed leaking at least once when milked once daily during the last final week of lactation, but no comparison was made with the abrupt cessation group.Moreover, for every 4.5-kg increase at the last test day yield above 18.1 kg, the odds of leakage increased by 31% in gradually dried off cows.After milking cessation, results are inconsistent with a study reporting no differences (Larsen et al., 2021), another that cows dried off gradually tended to leak more (Gott et al., 2016), and others reporting the opposite (Zobel et al., 2013;De Prado-Taranilla et al., 2020).Regarding udder health at calving, no differences were observed between gradual and abrupt milking cessation in SCC (Wieland et al., 2023) and IMI (Gott et al., 2017), but, although 1× milking was beneficial for primiparous cows, multiparous cows had increased odds of IMI at calving with gradual milking cessation compared with abrupt dry-off (Gott et al., 2016).It is important to consider that, during the week leading to dry-off, milk is still actively produced and might exacerbate the engorgement, resulting also in tissue damage and pain (Bertulat et al., 2013).At the same time, cows, in particular if high-producing, have a strong motivation to be milked, and suddenly changing their routine (i.e., abrupt milking interruption) could have negative implications on welfare.Zobel et al. (2013) observed that cows abruptly dried off had increased time standing at the gate at regular milking hours after dry-off, highlighting their motivation to be milked.Moreover, it would appear that high yield when dry-off is carried out abruptly increased udder firmness and pain (i.e., numerically higher probability of avoidance behavior to udder palpation) but there is currently limited evidence to support the practice of intermittent milking from a udder health perspective (Wieland et al., 2023).In automatic milking systems, it is also possible to tailor the process to each cow, setting different protocols of milking frequency paired or not with restrictions in concentrate allowance to allow for an actual gradual milking cessation (Martin et al., 2020;France et al., 2022).
Several dietary strategies with diversified intensities have been tested in recent years.Feed restriction can consist of a change in amount of ration provided (i.e., limiting the intake allowance) or a change in energy density of the diet (i.e., reducing concentrates inclusion in the diet, diluting the lactation TMR with straw, or feeding forages only; Leduc et al., 2021).The milk output reduction achievable depends on the severity of the restriction, varying from 40 to 60% (Figure 1).A reduced amount of energy (and even AA) available from the diet can lower yield in a few days (Larsen et al., 2021).Marked feed restriction also causes profound hormonal changes, with a decrease in circulating insulin, IGF-1, leptin, and prolactin, and an increase in growth hormone, progesterone, and cortisol concentrations, promoting a catabolic state and redirecting nutrients to vital organs (Leduc et al., 2021), similarly to what happens in early lactation.Moreover, the changes in IGF-1 and prolactin, together with the decreased mammary blood flow and nutrient uptake (Guinard-Flament et al., 2007), can be suggestive of an anticipated mammary involution, as supported by the changes noted in milk and mammary secretion composition (Ollier et al., 2014).However, if the restriction is too severe, cows show signs of hunger, increasing vocalizations (Tucker et al., 2009;Franchi et al., 2019), which suggests an impairment of welfare.In addition, cows that underwent a more marked feed restriction at dry-off had increased plasma cortisol, the primary marker of stress (Odensten et al., 2007).Moreover, the sudden decrease in energy intake depresses glucose availability, the main energy source of activated immune cells (Kvidera et al., 2017), and increases blood concentrations of BHB and NEFA (Ollier et al., 2014(Ollier et al., , 2015;;Jermann et al., 2022), which are known to have inhibitory effects on leukocytes (Minuti et al., 2020).In fact, PBMC proliferation and IL-4 production were reduced in cows fed only hay before dry-off (Ollier et al., 2014).An active and efficient immune system is pivotal in this phase, to promote mammary involution (Zhao et al., 2019) and prevent new IMI.
Long-term effects of feed restriction and milk volume reduction at dry-off on productivity in the ensuing lactation are still unclear.Gott et al. (2017) reported no association between milk yield and SCC in the subsequent lactation and once daily milking but, overall, higher amounts of milk produced at the final test day was associated higher SCC in the following lactation.Herve et al. (2019) did not observe any carryover effect on milk production at refeeding in mid-lactation cows that were feed restricted, despite an elevated rate of mammary epithelial cell exfoliation.Different types of stressors in the early dry period can have persistent effects.Heat stress in the early dry period can impair milk production in the subsequent lactation, as a result of a compromised mammary involution process (Fabris et al., 2019).Moreover, the effects of a certain degree of undernutrition in this stage of gestation on the offspring are unclear.Stressors during the early dry period are known to affect offspring performance.However, there is a lack of information on the effects of the transient nutrient restriction of the dam in late gestation on newborn dairy calves, both directly (through in utero effects) and indirectly (through colostrum).Thus, severe feed restriction (either qualitative or quantitative) in this phase can be a double-edged sword, with possible positive short-term effects on involution and udder engorgement but unknown long-term effects on the cow and calf.The combination of milking and feeding restriction showed the best results in terms of milk reduction in most of the studies reported in Figure 1, merging the effects of a moderate feed restriction and the reduction of milking frequency (Tucker et al., 2009;Larsen et al., 2021;France et al., 2022).These 2 strategies are additive, but they effectively reduce yield via different feedback mechanisms (Guinard-Flament et al., 2007).Feed restriction is associated with a reduction in glucose supply to the mammary gland, through a downregulation of gene expression of glucose transporters in mammary epithelial cells, whereas once daily milking decreases the expression of α-LA and κ-CN, decreasing milk and lactose synthesis and decreasing glucose uptake (Boutinaud et al., 2008).Cows milked 1× with a moderate feed restriction during the week preceding dry-off had a 10 kg (~40%) decrease in milk yield, paired with higher milk fat, protein, and SCC, and increased daily rumination time compared with the predrying period (Dancy et al., 2019).Interestingly, in this period they also had higher plasma glucose and lower BHB.Similar results were observed by Odensten et al. (2005).In their study, cows were milked 1× and fed either only straw or silage.During the dry-off period, both groups had higher milk fat and protein concentrations, lower milk lactose and plasma BHB before dry-off compared with the preceding days.Moreover, straw-fed cows had an increase in NEFA concentration, whereas silage-fed cows had only a moderate increase in NEFA and an increase in glycemia.These results suggested that reducing milking frequency decreased glucose mammary uptake, which was not compensated by a mild decrease in dietary energy, thus limiting the negative effects of undernutrition (e.g., BHB concentration).Other effects reported in Table 1, such as the changes in feeding behavior, hormonal status, and rumen fermentation, are likely more related to the feed restriction.
To summarize, the feeding approach might be more effective in terms of yield reduction but both methods have their side effects.With the feed reduction, yield is lowered by a decrease in nutrient availability to the mammary gland rather than by a mechanism that involves increasing udder pressure and reducing mammary glucose uptake.However, restricting intake increases feeding motivation and vocalizations, which are signs of hunger (Tucker et al., 2009;Franchi et al., 2019).On the other hand, feed restriction improved udder-related responses (i.e., reduced avoidance response to udder stimulations), indicating that reduced odds of experiencing pain or discomfort at the udder level come along with the reduced nutrient intake (Franchi et al., 2022).In contrast, milking once per day can cause milk leakage before dry-off (Gott et al., 2016), with a greater proportion of cows showing leakage compared with cows milked twice (Larsen et al., 2021), and can impair cow comfort.Nevertheless, if the feeding level is reduced together with milking, no differences were observed in udder engorgement and milk leakage after dry-off (Larsen et al., 2021), alongside the benefits on energy balance.
Other possible approaches include treatments targeted to inhibit prolactin secretion, promote involution, or depress feed intake.Treatments with acidogenic boluses, aimed at reducing intake (Maynou et al., 2018), did not show the ability to effectively reduce milk yield, whereas prolactin inhibitors and casein hydrolysate promoted mammary involution if infused before or at dry-off.Casein hydrolysate is a local regulator of mammary gland function and provokes the loss of tight junction integrity.Its infusion into the udder causes changes in mammary secretions similar to those caused by involution (increased levels of Na + and K + , immunoglobulins, and lactoferrin), promote a local inflammatory response, and had positive effects on IMI prevention and subclinical mastitis cure rates during the dry period and on milk production in the subsequent lactation (Shamay et al., 2003;Shoshani and van Straten, 2022).Prolactin is the hormone that promotes lactation, and its inhibition depresses milk synthesis.Daily single or double infusions of dopamine agonists (quinagolide or cabergoline), performed for a few days around dry-off, decreased prolactin and milk synthesis and likely promoted involution, as suggested by the changes in milk SCC, albumin, Na + -to-K + ratio, and citrate-to-lactoferrin ratio, which are markers of membrane integrity (Ollier et al., 2014).Moreover, the drop in milk yield was obtained with only a limited decrease in feed intake and without metabolic disturbances, promoting a protective effect against a Streptococcus agalactiae challenge (Ollier et al., 2015).A single injection of cabergoline at dry-off reduced also the risk of milk leakage, even though caused a drop in feed intake for approximately 24 h the day after the infusion (Larsen et al., 2021).Therefore, prolactin inhibitor infusions may be a viable solution to decrease milk yield and promote involution, despite some adverse effects on feed intake and feeding behavior that require further investigation (Larsen et al., 2021).However, it is not authorized in some parts of the world, including European Union, because of serious adverse events (including death) associated with the use.
Delaying dry-off to a time when milk production has declined can be an alternative way to obtain cows producing less milk, without the additional labor and costs of the other practices.There is a plethora of literature focusing on the effects of implementing a short or no dry period (van Knegsel et al., 2013;Kok et al., 2019), but results of those studies are inconsistent with regard to metabolic, health, and behavioral effects.Another option to extend the lactation is to increase the voluntary waiting period for first insemination (van Knegsel et al., 2022), particularly in the case of animals known to have a persistent lactation curve; an approach that could result in lower milk production at the time of dry-off (Burgers et al., 2021).Multiparous cows suitable for a longer lactation could be identified based on previous lactations, whereas, in primiparous (that usually have greater persistence than multiparous), early lactation performances might be used as selection criteria (Lehmann et al., 2017).

CONCLUSIONS
Dry-off is a critical event of the lactation cycle and sets the stage for a healthy dry period.The average milk yield at dry-off has increased in recent years and represents a threat to positive outcomes of the dry period.Several methods have been proposed to reduce production before milking cessation, including feed restriction, gradual milking, and prolactin inhibitors.All the proposed strategies can provide benefits for the cows, but they concurrently entail side effects.Nevertheless, reducing milk yield before dry-off appears to be necessary for cows with high production at the end of lactation (over 15 kg/d), and particular care should be given when applying selective DCT approaches.Therefore, developing additional and alternative ways to dry-off high-yielding cows, without side effects, is still needed.To fully assess their effectiveness, characterization of the metabolic, inflammatory, and behavioral (i.e., welfare) responses is required.
Figure 1.Decrease in milk yield (%) achieved from the beginning of the dry-off procedure to the day before the last milking with different strategies (MILK = reduction in milking frequency; FEED = feed or nutrient restriction; MILK × FEED = reduction in milking frequency paired with changes in feeding; PRL = 1 or 2 injections/d of prolactin inhibitor, before dry-off) in studies available in the literature involving dairy cows with milk yield higher than 15 kg/d at the beginning of treatments.*Reduction achieved at the last milking after a day of no milking.

Table 1 .
Cattaneo et al.: LITERATURE REVIEW: DRY-OFF HIGH-YIELDING DAIRY COWS Effects of different methods to reduce milk yield before dry-off on DMI, behavior, milk composition, blood hormones, and metabolites in high-yielding dairy cows 1 Milk = reduction in milking frequency; feed = feed or nutrient restriction; milk × feed = reduction in milking frequency paired with changes in feeding.